1

20 Applications of PHAs in Medicine and Pharmacy

Dr. Simon F. Williams1, Dr. David P. Martin2 1 Tepha, Inc. 303 Third Street, Cambridge, MA 02142, U.S.A.; Tel.: ‡01-617-492-0505; Fax: ‡01-617-492-1996; E-mail: [email protected] 2 Tepha, Inc. 303 Third Street, Cambridge, MA 02142, U.S.A.; Tel.: ‡01-617-492-0505; Fax: ‡01-617-492-1996; E-mail: [email protected]

1 Introduction ...... 3

2 Historical Outline ...... 3

3 PHA Preparation and Properties: A Primer ...... 5 3.1 Production ...... 5 3.2 Mechanical and Thermal Properties ...... 5 3.3 Sterilization of PHA Polymers ...... 6

4 Biocompatibility ...... 7 4.1 Natural Occurrence ...... 7 4.2 In Vitro Cell Culture Testing ...... 8 4.3 In Vivo Tissue Responses ...... 9

5 Biodistribution ...... 11

6 Bioabsorption ...... 11 6.1 In Vitro Degradation ...... 11 6.2 In Vivo Bioabsorption ...... 12

7 Applications ...... 13 7.1 Cardiovascular ...... 13 7.1.1 Pericardial Patch ...... 13 7.1.2 Artery Augmentation ...... 14 7.1.3 Atrial Septal Defect Repair ...... 15 7.1.4 Cardiovascular Stents ...... 15 7.1.5 Vascular Grafts ...... 15 2 20 Applications of PHAs in Medicine and Pharmacy

7.1.6Heart Valves ...... 16 7.2 Dental and Maxillofacial ...... 17 7.2.1 Guided Tissue Regeneration ...... 17 7.2.2 Guided Bone Regeneration ...... 18 7.3 Drug Delivery ...... 18 7.3.1 Implants and Tablets ...... 19 7.3.2 Microparticulate Carriers ...... 20 7.4 Prodrugs ...... 24 7.5 Nerve Repair ...... 24 7.6Nutritional Uses ...... 24 7.6.1 Human Nutrition ...... 24 7.6.2 Animal Nutrition ...... 25 7.7 Orthopedic ...... 25 7.8 Urology ...... 26 7.9 Wound Management ...... 26 7.9.1 Sutures ...... 26 7.9.2 Dusting Powders ...... 27 7.9.3 Dressings ...... 27 7.9.4 Soft Tissue Repair ...... 27

8 Future Directions ...... 27

9 Patents ...... 28

10 References ...... 30

HA hydroxyapatite mcl-PHA medium chain-length PHA

Mw molecular weight PCL polycaprolactone PGA polyglycolic acid PHA polyhydroxyalkanoate PLA polylactic acid poly=3HB)) poly-R-3-hydroxybutyrate poly=3HB-co-3HV)) poly-R-3-hydroxybutyrate-co-R-3-hydroxyvalerate poly=3HB-co-4HB)) poly-R-3-hydroxybutyrate-co-4-hydroxybutyrate poly=3HO-co-3HH)) poly-R-3-hydroxyoctanoate-co-R-3-hydroxyhexanoate poly=3HP)) poly-3-hydroxypropionate poly=4HB)) poly-4-hydroxybutyrate Poly=5HV)) poly-5-hydroxyvalerate poly=6HH)) poly-6-hydroxyhexanoate

Tg glass transition temperature

Tm melting temperature 2 Historical Outline 3

1 addition, the PHA polymers are thermo- Introduction plastic in nature, with a wide range of thermal properties, and can be processed Polyhydroxyalkanoates =PHAs) are a class of using conventional techniques =Holmes, naturally occurring polyesters that are pro- 1988). duced by a wide variety of different micro- organisms =Steinbüchel, 1991). Although they are derived biologically, the structures 2 of these polymers bear a fairly close resem- Historical Outline blance to some of the synthetic absorbable polymers currently used in medical applica- As a class of polymers, the PHAs are relative tions. Owing to their limited availability, the newcomers, with many of the different types PHAs have remained largely unexplored, yet having been discovered during only the past these polymers offer an extensive range of 20 years. One of the simplest members of the properties that extend far beyond those class, poly-R-3-hydroxybutyrate, poly=3HB), currently offered by their synthetic counter- is an exception as it was first identified in parts. 1925 and is the most well-known PHA At the last count there were well over 100 polymer. It should be noted however, that different types of hydroxy acid monomers the properties of poly=3HB) are not repre- that had been incorporated into PHA poly- sentative of the polymer class as a whole. mers, and the list is continuing to grow During the 1980s, the British company, =Steinbüchel and Valentin, 1995). These Imperial Chemical Industries =ICI), devel- monomers include hydroxyalkanoate units oped a commercial process to produce po- ranging from 2- to 6-hydroxy acids substi- ly=3HB), and a related copolymer known tuted with a wide range of groups including as poly-R-3-hydroxybutyrate-co-R-3-hydroxy- alkyl, aryl, alkenyl, halogen, cyano, epoxy, valerate, poly=3HB-co-3HV). These poly- ether, acyl, ester, and acid groups =see mers were sold under the tradename of Figure 1). By no means will all of these Biopol, and were developed primarily as monomers be useful or suitable for medical renewable and biodegradable replacements use; however, they provide a set of materials for petroleum-derived plastics. As a result of with properties that range from rigid and these activities and others =Lafferty et al., stiff to flexible and elastomeric, including 1988), both polymers became widely avail- polymers that degrade relatively quickly in able, which in turn provided opportunities vivo and others that are slow to degrade. In for their evaluation as medical biomaterials. While these efforts have resulted in several promising clinical trials, and development R O efforts continue, products containing these materials have yet to be approved for in vivo CH2 O x medical use. n In 1993, ICI transferred its biological Typical values of x, n and R division to Zeneca, which continued to x = 1 to 4 develop PHAs for commodity applications n = 1,000 to 10,000 under the tradename Biopol. Zeneca, how- R = alkyl group (CmH2m+1) or functionalized alkyl group ever, sold its Biopol assets to Monsanto in the Fig. 1 General chemical structure of the PHAs. mid-1990s. In 2001, an American company, 4 20 Applications of PHAs in Medicine and Pharmacy

Metabolix, Inc. acquired the Biopol assets range of tissue-engineered products based from Monsanto, and is developing trans- on PHA polymers, and is expanding the genic approaches to the large-scale manu- number available for medical research to facture of PHAs through fermentation and meet both the needs of tissue engineering agricultural biotechnology. and the development of more traditional More recent interest in the use of PHA medical devices. As a result of these efforts polymers for medical applications has arisen during the past two years, the number of primarily in response to the needs of the materials currently under evaluation has emerging field of tissue engineering, where expanded and now includes three additional a much wider range of absorbable polymers PHA polymers, namely, poly-R-3-hydroxy- are being sort for use as tissue scaffolds. In octanoate-co-R-3-hydroxyhexanoate =poly=3- fact, in the past two years PHAs have become HO-co-3HH)), poly-4-hydroxybutyrate =poly- one of the leading classes of biomaterials =4HB)), and poly-R-3-hydroxybutyrate-co-4- under investigation for the development of hydroxybutyrate =poly=3HB-co-4HB)). This tissue-engineered cardiovascular products brings the total number of PHA polymers because they can offer properties not avail- currently under investigation for medical able in existing synthetic absorbable poly- application to five =Figure 2). mers. An American company, Tepha, Inc., is currently engaged in the development of a

O CH3 Poly(3HB) O n

O CH3 O C2H5 Poly(3HB-co-3HV) O O n m

Poly(4HB) O O

n

O CH3 O O Poly(3HB-co-4HB) O n m

O C5H11 O C3H7 Poly(3HO-co-3HH) O O Fig. 2 Chemical structures of PHAs currently n m under medical investigation. 3 PHA Preparation and Properties: A Primer 5

3 500,000, although PHAs with much longer PHA Preparation and Properties: A Primer pendant groups =known as medium chain- length PHAs, mcl-PHAs), such as po-

3.1 ly=3HO-co-3HH), typically have Mw that are Production closer to 100,000. Polydispersity is typically around 2.0. By isolating the enzymes re- The PHA polymers are accumulated as sponsible for PHA production, namely PHA discrete granules within certain microorgan- synthases, researchers have also been able to isms at levels reaching 90% of the dry cell produce PHA polymer in vitro with ultra- mass, and can be isolated fairly readily by high Mw exceeding several million =Gern- breaking open the cells and using either an gross and Martin, 1995), and also in vivo in aqueous-based or solvent-based extraction transgenic organisms =Kusaka et al., 1997; process to remove cell debris, lipid, nucleic Sim et al., 1997). acids, and proteins. Traditionally, these polymers have been produced by fermenta- 3.2 tion from sugars or oils, often with co-feeds, Mechanical and Thermal Properties and the majority of medical studies on poly=3HB), poly=3HB-co-3HV), and po- As a class of polymers, the PHAs offer an ly=3HO-co-3HH), have been based on poly- extensive design space with properties span- mers derived via this route. ning a large range, and usefully extending During the late 1980s, the genes respon- the relatively narrow property range offered sible for PHA production were isolated, and by existing absorbable synthetics =Engelberg this has led more recently to the develop- and Kohn, 1991). The mechanical properties ment of transgenic methods for PHA pro- of the five PHAs currently being investigated duction =see Williams and Peoples, 1996, for medical use are shown in Table 1. The and references therein). This breakthrough homopolymer, poly=3HB), is a relatively has provided a new means of tailoring the stiff, rigid material that has a tensile strength properties of PHA polymers to particular comparable with that of polypropylene. The applications, and represents a potentially introduction of a comonomer into this important advance in the development of polymer backbone, however, significantly technologies to produce designer biomate- increases the flexibility and toughness of rials for medical use. Poly-4-hydroxybutyrate the polymer =extension to break and impact =poly=4HB)), for example, is produced using strength), and this is accompanied by a this technology. Transgenic PHA production reduction in polymer stiffness =Young's may also prove to be important in the modulus). This is evident in the poly=3HB) medical field from a regulatory standpoint, copolymers, poly=3HB-co-3HV) and po- since this technology allows the production ly=3HB-co-4HB) =Doi, 1990; Sudesh et al., host to be selected. For example, PHAs may 2000). now be produced by fermentation in Esche- A progressive and substantial change in richia coli K12, a well-characterized host used the mechanical properties of poly=3HB) also extensively by the biotechnology industry. occurs when the pendant groups are ex- In general, PHA polymers are produced tended from the polymer backbone. The with relatively high molecular weights =Mw) mcl-PHA, poly=3HO-co-3HH), for example, in vivo. Commercial grades of poly=3HB) shares the same backbone as poly=3HB), but copolymers typically have Mw that are at least in contrast is a highly flexible thermoplastic 6 20 Applications of PHAs in Medicine and Pharmacy

elastomer with properties comparable with cessed. Tg values are also depressed by the those of commercially produced materials incorporation of monomers with longer =Gagnon et al., 1992). pendant groups, the depression being rela- Extending the distance between the ester tively modest in the poly=3HB) copolymers, groups in the PHA backbone can also have a but pronounced for the mcl-PHAs. dramatic impact on mechanical properties. The homopolymer poly=4HB), for example, is a highly ductile, flexible polymer with an 3.3 extension to break of around 1000%, com- Sterilization of PHA Polymers pared with poly=3HB), which has an exten- sion to break of less than 10%. Combining For medical use, most PHAs have been these different monomers to form copoly- sterilized using ethylene oxide, without mers, as in poly=3HB-co-4HB), produces one causing any significant changes to the series of materials with a wide range of physico-chemical properties of the poly- useful mechanical properties that can be mers. However, low-melting PHAs such as tailored to specific needs. Interestingly, at poly=4HB) and poly=3HO-co-3HH) are gen- levels of around 20 ± 40% 4HB, the po- erally sterilized using a cold cycle, particu- ly=3HB-co-4HB) copolymers actually behave larly if the polymer has been fabricated ready like elastic rubbers. for use. Residual ethylene oxide levels in The thermal properties of PHAs also span poly=3HO-co-3HH) after cold sterilization wide ranges =see Table 1). Typical melting with ethylene oxide for 8 h at 38 8C with 65%

temperatures =Tm) range from around 55 8C humidity have been reported to be <1 ppm for poly=3HO-co-3HH) to about 180 8C for after one week =Marois et al., 1999a). the poly=3HB) homopolymer. Glass transi- Several studies have described the effects

tion temperatures =Tg) span the range from of g-irradiation on PHA polymers derived

about ±55 8C to about 5 8C. In general, Tm from 3-hydroxy acids, such as poly=3HB), values decrease as the pendant groups poly=3HB-co-3HV), and poly=3HO-co-3HH). become longer. This is particularly impor- It has been reported that poly=3HB), unlike tant in the melt processing of poly=3HB), polyglycolic acid =PGA), can be sterilized by which is unstable at temperatures just above g-irradiation doses on the order of 2.5 Mrad its melting point. Incorporation of other =Holmes, 1985), although it is likely that monomers into the poly=3HB) polymer some reduction in molecular weight results backbone yields lower-melting poly=3HB) from this treatment. At higher doses =10 ± copolymers that can be more readily pro- 20 Mrad) the mechanical integrity of both

Tab. 1 Mechanical and thermal properties of some representative PHAs

PHA Poly- Poly3HB-co- Poly4HB) Poly3HB-co- Poly3HO-co- 3HB) 20%3HV) 16%4HB) 12%3HH)

Melting temperature [Tm, 8C] 177 145 53 152 61

Glass transition temperature [Tg, 8C] 4 ±1 ±48 ±8 ±35 Tensile strength [MPa] 40 32 104 269 Tensile modulus [GPa] 3.5 1.2 0.149 n.d. 0.008 Elongation at break [%] 650 1000 444 380 4 Biocompatibility 7 poly=3HB) and poly=3HB-co-3HV) are sig- cellular debris in industrial samples of nificantly compromised =Miller and Wil- poly=3HB-co-3HV), Rouxhet et al. =1998) liams, 1987). Luo and Netravali =1999) have detected a number of contaminants on the also reported significant changes in the surface of these samples by X-ray photo- mechanical properties and Mw of po- electron spectroscopy, and Williams et al. ly=3HB-co-3HV) after exposure to g-irradia- =1999) reported that an industrial sample of tion at doses of 10 ± 25 Mrad. poly=3HB) contained more than 120 endo- Exposure of poly=3HO-co-3HH) to g-irra- toxin units per gram. Two methods to diation at a dose of 2.5 Mrad at room remove endotoxin have been reported re- temperature has also been reported to result cently, one being based primarily on the use in a loss of molecular weight on the order of of peroxide =Williams et al., 1999) and the 17%; this is caused by random chain other by use of sodium hydroxide =Lee et al., scission, accompanied by some degree of 1999). physical cross-linking =Marois et al., 1999a). Thus, while g-irradiation is generally recog- 4.1 nized as a desirable alternative to ethylene Natural Occurrence oxide for sterilization, care must be exercised in its use on PHA polymers, and the Some of the monomers incorporated into procedures carefully validated. PHA polymers are known to be present in A few PHA polymers may also be steri- vivo, and both their metabolism and excre- lized by steam =Baptist and Ziegler, 1965), tion are well understood. The monomeric particularly if they have Tm over 140 8C, and component of poly=3HB), R-3-hydroxybuta- are thermally stable at this temperature. noic acid, for example, is a normal metab- Holmes =1985) has reported that poly=3HB) olite found in human blood. This hydroxy powders can be sterilized in this manner. acid is a ketone body, and is present at concentrations of 3 ± 10 mg per 100 mL blood in healthy adults =Hocking and Mar- 4 chessault, 1994). This monomer has been Biocompatibility administered to obese patients undergoing therapeutic starvation to reduce protein loss Without doubt, the biological response to =Pawan and Semple, 1983), and also eval- PHA polymers in vivo represents the most uated as an intravenously administered important property of these biomaterials if a energy source in both humans =Hiraide medical application is being contemplated. and Katayama, 1990) and piglets =Tetrick Most of the information currently available et al., 1995). There is also interest in the use relates to poly=3HB) and poly=3HB-co-3HV), of this monomer in ocular surgery as an and has been recently reviewed =Hasirci, irrigation solution to maintain the tissues 2000). A small amount of information on =Chen and Chen, 1992). poly=3HO-co-3HH), poly=3HB-co-4HB), and The monomeric component of poly=4HB), poly=4HB) has also been published. Care 4-hydroxybutanoic acid, is also a naturally should be exercised in interpreting these occurring substance that is widely distrib- data however, since most studies have been uted in the mammalian body, being present based on the use of industrial rather than in the brain, kidney, heart, liver, lung, and medical grades of PHA polymers. Notably, muscle =Nelson et al., 1981). This 4-hydroxy Garrido =1999) has described the presence of acid has been used for over 35 years as an 8 20 Applications of PHAs in Medicine and Pharmacy

intravenous agent for the induction of an in vitro cell culture test method with anesthesia and for long-term sedation =Ent- mouse fibroblasts, and reported that the holzner et al., 1995). It is also one of the most extract appeared slightly to suppress cellular promising treatments for narcolepsy =Scharf activity. et al., 1998), although unfortunately as with In other in vitro testing, Rivard et al. =1995) many hypnotics there has been some illegit- showed that porous poly=3HB-co-9%3HV) imate use of this compound. However, since substrates =Selmani et al., 1995), when seed- the half-life of the acid is short =35 min), and ed with canine anterior cruciate ligament relatively high doses =several grams) are =ACL) fibroblasts, sustained a cell prolifer- required to obtain any hypnotic effect, small ation rate similar to that observed in collagen implants of poly=4HB) could not induce sponges for around 35 days, with maximal general sedation, for example. cell density occurring after 28 days. Interest- In addition to the known presence of ingly, the poly=3HB-co-9%3HV) substrates certain PHA monomers in humans, low maintained their structural integrity during molecular-weight forms of poly=3HB) have the culturing, whereas the collagen foams also been detected in human tissues. Reusch contracted substantially and produced sig- and colleagues first identified poly=3HB) in nificantly less protein. In evaluating po- blood serum =0.6± 18.2 mg L À1) complexed ly=3HB) as a potential drug delivery matrix, with low-density lipoproteins, and with the Korsatko et al. =1983a) also reported no carrier protein albumin =Reusch et al., significant differences in cellular growth 1992). The oligomers have also been detect- with mice fibroblasts. ed in human aorta =Seebach et al., 1994), and Saito et al. =1991) evaluated poly=3HB) are known to form ion channels in vivo when sheets in an inflammatory test using the complexed with polyphosphate =Reusch chorioallantoic membrane of the developing et al., 1997). egg, and reported that the polymer did not cause any inflammation. 4.2 Several reports have described the effects In Vitro Cell Culture Testing of small, low molecular-weight, crystalline particles of poly=3HB) on the viability of Relatively few studies have attempted to cultured macrophages, fibroblasts, co-cul- characterize the tissue response of PHAs tures of Kupffer cells and hepatocytes, and caused by leachables such as impurities, osteoblasts =Ciardelli et al., 1995; Saad et al., additives, monomers, and degradation prod- 1996a,b,c). These particles represent one of ucts. Chaput et al. =1995) evaluated the the degradation products expected to arise in cytotoxic responses of three poly=3HB-co- vivo from the absorption of poly=3HB) and 3HV) compositions =7, 14, and 22% hydroxy- DegraPol, a phase-segregated multiblock valerate) using direct contact and agar polyesterurethane copolymer. At low con- diffusion cell culture tests, and reported that centrations, the small poly=3HB) particles the solid polymers elicited mild to moderate were found to be well tolerated by macro- cellular reactions in vitro. However, the phages, fibroblasts, Kupffer cells and hepa- cytotoxicity of extracts from these polymers tocytes. Macrophages, Kupffer cells, and to a varied with the medium, surface-to-volume lesser extent fibroblasts and osteoblasts, ratio, time and temperature. Dang et al. were all found to take up =phagocytose) the =1996) also evaluated an extract from an small particles of poly=3HB) =1 ± 20 mm), and industrial sample of poly=3HB-co-3HV) in evidence of biodegradation by macrophages 4 Biocompatibility 9 was also found =Ciardelli et al., 1995). He- bated in a bioreactor under pulsatile flow. patocytes, in contrast, demonstrated no After eight days, near-confluent layers of signs of poly=3HB) phagocytosis. At high cells were observed with the formation of concentrations = > 10 mgmLÀ1), phagocyto- extracellular matrix. Sodian et al. =2000a) sis of poly=3HB) particles was found to cause also studied cellular attachment to porous cell damage and cell activation in macro- samples of poly=4HB), and compared the phages and to a lesser degree in osteoblasts, results to those obtained with a porous but not in fibroblasts =Saad et al., 1996a,b,c). poly=3HO-co-3HH) material and a PGA Separately, the chondrocyte compatibility of mesh. After seeding and incubating these a DegraPol foam was also evaluated in vitro. materials with ovine vascular cells for eight Rat chondrocytes were found to attach to days, there were significantly more cells on about 60% of the foam compared with a the PGA, although after exposure to flow no polystyrene control, and proliferated at com- significant differences were found. A con- parable rates =Saad et al., 1999), leading to siderable amount of collagen development the conclusion that the DegraPol foam had was noted for each sample, with the highest acceptable chondrocyte compatibility. amounts present in the PGA meshes. Cel- Of particular interest in the evolving field lular attachment to a composite of po- of tissue engineering was a report by ly=4HB) with a PGA mesh has also been Rouxhet et al. =1998) on the effect of adhe- evaluated in vitro recently, and compared sion and proliferation of monocytes-macro- with the mesh alone and a poly=4HB) foam phages to a poly=3HB-co-8%3HV) film when =Nasseri et al., 2000). Better cell migration modified by hydrolysis or coated with differ- into the composite, and better shape reten- ent proteins. As anticipated, the cells were tion were observed. found to have a greater affinity for the polymer surface after it had been hydrolyzed 4.3 to liberate additional carboxylate and hydrox- In Vivo Tissue Responses yl functions. However, it was also found that adhesion of this cell type increased signifi- Some of the earliest investigations of the in cantly when fibronectin was adsorbed to the vivo tissue responses to PHA polymers were polymer surface, but not when collagen or made by W. R. Grace and Co. in the mid- albumin were pre-absorbed. 1960s =Baptist and Ziegler, 1965). In these Cellular attachment to porous tubes made early studies, film strips of poly=3HB) were from poly=3HO-co-3HH) under different implanted subcutaneously and intramuscu- seeding conditions has been evaluated larly in rabbits, and removed after eight =Stock et al., 1998). Although dynamic cell weeks. Examination of the implant sites seeding techniques were found initially to revealed granulomatous foreign body reac- result in a higher rate of ovine smooth tions, but these did not affect the underlying muscle cellular attachment compared with area. static seeding, higher attachment was not Since these early investigations, many sustained under simulated blood flow con- reports have been made describing the in ditions. Cell attachment to a composite vivo tissue responses of poly=3HB) and material of PGA and poly=3HO-co-3HH) poly=3HB-co-3HV) in both biocompatibility has also been reported =Sodian et al., 1999). and application-directed studies. Chaput After seeding with myofibroblasts and en- et al. =1995) described one of the longest in dothelial cells, these composites were incu- vivo studies, in which poly=3HB-co-3HV) 10 20 Applications of PHAs in Medicine and Pharmacy

films =containing 7, 14, and 22% valerate) gen deposition following an in vivo study of were sterilized by ethylene oxide and im- subcutaneous poly=3HB) implants in rats. planted intramuscularly in sheep for up to Williams et al. =1999) reported a 40-week 90 weeks. No abscess formation or tissue subcutaneous implant study of poly=3HO-co- necrosis was seen in the vicinity of the 3HH) in mice. At two weeks, there was implants. However, after 1 week in vivo, minimal reaction to the implants which had acute inflammatory reactions with numer- been encapsulated by a thin layer of fibro- ous macrophages, neutrophils, lymphocytes blasts, four to six cell layers thick, surround- and fibrocytes were observed in a capsule at ed by collagen. There was no evidence of the interface between the polymers and the macrophages, and the tissue response con- muscular tissues. After 11 weeks, the ob- tinued to be very mild at 4, 8, 12 and 40 served reaction was less intense with a lower weeks, with the amount of connective tissue density of inflammatory cells present, surrounding the implants remaining fairly though lymphocytes were still observed in constant. The polymer proved to be partic- the capsule and muscular tissues. At this ularly inert, and could be readily removed stage, the capsules were reported to consist with little tissue adherent to the implants. primarily of connective tissue cells, and were An extract from poly=3HO-co-3HH) was also dense and well-vascularized with highly tested in a standard skin sensitization test organized oriented fibers and fibroblastic =ASTM F270), but no discernable erythema/ cells aligned in parallel with the polymer eschar formation was observed. surfaces. A large number of fatty cells were Subcutaneous implants of poly=4HB) also observed in the capsule, as well as at the have also been reported to be well tolerated interface and in adjacent muscles after long- in vivo during the course of their degradation term implantation =at 70 and 90 weeks). =Martin et al., 1999), with minimal inflam- Interestingly, few differences were observed matory responses occurring. between the capsules, tissue characteristics It is worth noting that, in vivo, as most or cellular activity in terms of the composi- PHA polymers break down they release tions of the three poly=3HB-co-3HV) poly- hydroxy acids that are significantly less mers. acidic and less inflammatory than many Similar results were also observed by currently used synthetic absorbable poly- Gogolewski et al. =1993) when poly=3HB) mers =Taylor et al., 1994). For example, and poly=3HB-co-3HV) samples were im- poly=3HB) and poly=4HB), are derived from

planted subcutaneously in mice. Fibrous 3- and 4-hydroxybutanoic acids =pKa 4.70 and capsules of around 100 mm thickness devel- 4.72, respectively), that are significantly less oped after one month, and these increased to acidic than the 2-hydroxy acids =glycolic acid,

200 mm by three months, but then thinned to pKa 3.83; lactic acid, pKa 3.08) found in PGA 100 mm at six months. However, the number and poly-lactic acid =PLA). Furthermore, of inflammatory cells was found to increase significant differences in the mechanism of with valerate content, and a few granulocytes degradation of these synthetic polymers, were still present around blood vessels near which can degrade autocatalytically from encapsulated implants containing 22% val- the inside outward, can result in substantial erate at six months. Separately, Tang et al. amounts of acidic degradation products =1999) suggested that leachable impurities being released. In one clinical study, for and low molecular-weight poly=3HB) are at example, around 5% of the patients receiv- least partly responsible for increased colla- ing PGA screws had an inflammatory 6 Bioabsorption 11 reaction to the implants that was sufficient to marily upon their chemical compositions. warrant operative drainage =Böstman, 1991). Other factors such as their location, surface Finally, Holmes =1988) has reported that area, physical shape and form, crystallinity, poly=3HB) shows negligible oral toxicity, the species, and molecular weight can also be À1 LD50 being greater than 5 g kg . very important. While useful information can be derived from in vitro studies, results of in vitro studies with PHA polymers are not 5 always good indicators of in vivo behavior. Biodistribution 6.1 The biodistribution of poly=3HB) micro- In Vitro Degradation spheres in mice =Bissery et al., 1984a), and poly=3HB) granules in rats =Saito et al., In order to investigate the mechanism of 1991) has been investigated using 14C-label- degradation of poly=3HB) and poly=3HB-co- ing and, as anticipated, results have been 3HV) in vivo, a number of studies have been found to depend upon particle size. In the conducted to determine their rates of hy- first study, microspheres of 1 ± 12 mm diam- drolysis in vitro =see Holland et al., 1987, eter were injected intravenously into mice, 1990; Yasin et al., 1990; Knowles and Hast- and traced at 0.5, 1, and 24 h, and every seven ings, 1992; Chaput et al., 1995). These days thereafter. After 30 min, 47% of the studies have used complementary tech- radioactivity was found in the lungs, 14% in niques such as gravimetric and molecular the liver, and 2.1% in the spleen. After 1 h, weight analysis, as well as measurements of concentrations in the lungs and liver had surface and tensile properties to monitor increased to 62 and 16%, respectively, and by different aspects of degradation and develop 24 h there was still 60% in the lungs and a concept of the overall degradation process. 24% in the liver. Thereafter, the amounts This has led to the following general scheme remained fairly constant, but fell somewhat of in vitro behavior for poly=3HB) and in the lungs. In the rat study, granules of poly=3HB-co-3HV). Initially, some surface 500 ± 800 nm diameter were injected modification is observed, with water diffus- through a tail vein into rats and traced at ing into the polymer and porosity increasing. intervals of 2.5 h, 1 day, 13 days, and 2 Crystallinity also increases, but there is months. After 2.5 h, approximately 86% of relatively little change in molecular weight the radioactivity had accumulated in the in the first few months, and tensile proper- liver, with 2.5% and 2.4% of the total ties remain fairly constant. As the porosity distributed in the spleen and lungs, respec- increases, hydrolysis of the polymer chains tively. During the following two months, releases degradation products that can dif- radioactivity levels in most of the tissues fuse away more easily. The molecular weight decreased slowly, but steadily. decreases, erosion increases, and both weight and tensile strength begin to decrease more rapidly. At about one 1 year, the initial 6 resistance to degradation is followed by an Bioabsorption accelerated degradation with the material becoming more brittle, but not losing its The rates of bioabsorption of PHA polymers physical integrity. After one year, the most in vivo vary considerably, and depend pri- apparent change in the physical appearance 12 20 Applications of PHAs in Medicine and Pharmacy

of the polymer is loss of surface gloss and the this time period, no significant surface or development of surface rugosity. chemical modifications were observed, and Other in vitro studies have examined the no significant weight loss was detected. It action of additives such as polysaccharides was concluded that this polymer, which =Yasin et al., 1989), polycaprolactone =PCL) shares a common backbone with poly=3HB), =Yasin and Tighe, 1992), as well as lipases, degrades slowly by chemical hydrolysis. PHA depolymerases, and several extracts on Degradation of poly=4HB) in vitro has PHA degradation. Although PHA depoly- recently been reported =Martin et al., 1999). merases are abundant in the environment The homopolymer is fairly resistant to and are responsible for PHA biodegradation hydrolysis at pH 7.4, and over a 10-week in soil, there is currently no evidence that period very little degradation was observed, these enzymes are present in vivo. Mukai although a 20 ± 40% reduction in average et al. =1993) investigated the action of 16 molecular mass did occur during this time lipases on five different PHA polymers period. prepared either by fermentation or syntheti- cally, and found that none of these enzymes 6.2 catalyzed the hydrolysis of poly=3HB). How- In Vivo Bioabsorption ever, the other four PHA polymers were hydrolyzed by lipases, with the number of In early studies, some confusion arose lipases capable of hydrolyzing the PHA around the stability of poly=3HB) and po- polymer chains decreasing in the following ly=3HB-co-3HV) in vivo. Korsatko et al. order: poly-3-hydroxypropionate =poly- =1983a, 1984) and Wabnegg and Korsatko =3HP)) > poly=4HB) > poly-5-hydroxyvale- =1983) evaluated poly=3HB) for use as matrix rate =poly=5HV)) > poly-6-hydroxyhexa- retard tablets and reported that the polymer noate =poly=6HH)). Interestingly, two lipas- was degraded in vivo at a rate directly es have been detected recently in tissue proportional to the elapsed time =a zero- adjacent to poly=3HB) implants in rats, order reaction). However, it was reported raising the possibility of their involvement later that monofilaments derived from po- in poly=3HB) bioabsorption =Löbler et al., ly=3HB) and poly=3HB-co-3HV) =8 and 17% 1999). The copolymer, poly=3HB-co-3HV), valerate) showed little, if any, loss of strength formulated as microspheres with PCL, and when implanted subcutaneously in rats for loaded with bovine serum albumin, has also up to six months =Miller and Williams, been incubated with four different extracts in 1987), except after g-irradiation. Many sub- vitro =Atkins and Peacock, 1996a). The sequent studies have confirmed that po- percentage weight loss decreased in the ly=3HB) and poly=3HB-co-3HV) do degrade order newborn calf serum > pancreatin > in vivo, albeit slowly =Hasirci, 2000). Typi- synthetic gastric juice > Hanks' buffer, and cally, poly=3HB) is completely absorbed in it was speculated that the enhanced bio- vivo in 24 ± 30 months =Malm et al., 1992b; degradation in newborn calf serum, and Hazari et al., 1999a). During the first four surface erosion in pancreatin, must be due to weeks in vivo, the degree of crystallinity of a enzymatic activity in these extracts. sample of poly=3HB) implanted in the The in vitro degradation of poly=3HO-co- peritoneal cavity was reported to have in- 3HH) has also been examined for up to 60 creased, presumably as a result of the days =Marois et al., 1999b). When exposed to amorphous regions of the polymer degrad- acid phosphatase and b-glucuronidase for ing more rapidly than the crystalline do- 7 Applications 13 mains =Behrend et al., 2000a). After four ables, such as PGA. This might be advanta- weeks, crystallinity, Young's modulus, and geous, for example, in tissue regeneration microhardness were each shown to have applications where a sudden loss of a decreased fairly steadily, this being consis- mechanical property is undesirable, or more tent with a surface process. gradual loss of implant mass and steady in Kishida et al. =1989) attempted to develop a growth of new tissue are beneficial. method to accelerate the bioabsorption of poly=3HB) and poly=3HB-co-3HV) in vivo by adding basic compounds to the polymers. 7 Although in vitro the rate of hydrolysis was Applications found to increase, the effect in vivo was minimal, presumably because the basic Until recently, only poly=3HB) and po- accelerators had leached out. ly=3HB-co-3HV) were available commercial- The mcl-PHA, poly=3HO-co-3HH), also ly, and consequently the majority of inves- degrades slowly in vivo. Williams et al. =1999) tigations into applications have focused on a reported that the molecular weight =Mw)of relatively narrow set of polymer properties subcutaneous implants of poly=3HO-co- within the PHA design space. This situation 3HH) in mice decreased from 137,000 at is beginning to change however, with more implantation to around 65,000 over 40 recent studies involving poly=3HO-co-3HH), weeks, and that there were no significant poly=4HB) and poly=3HB-co-4HB). differences between the molecular weights of samples taken from the surfaces and 7.1 interiors of the implants. The latter finding Cardiovascular suggests that slow, homogeneous hydrolytic breakdown of the polymer occurs. Without doubt, the major medical use of While poly=3HB), poly=3HB-co-3HV), and PHAs has been in the development of poly=3HO-co-3HH) are generally degraded cardiovascular products. slowly in vivo, consistent with in vitro observations, the homopolymer, poly=4HB) 7.1.1 is an exception. Martin et al. =1999) found Pericardial Patch the in vivo degradation of this polymer to be One of the most advanced applications of relatively rapid, and to vary with porosity. PHA polymers in cardiovascular products Over a 10-week period it was reported that has been the development of a regenerative film, 50%, and 80% porous samples im- poly=3HB) patch that can be used to close the planted subcutaneously in rats, lost 20%, pericardium after heart surgery, without 50%, and nearly 100% of their mass, formation of adhesions between the heart respectively. The average molecular mass and sternum =Bowald and Johansson, 1990; of the polymer also decreased significantly, Malm et al., 1992a,b; Bowald and Johans- but independently of sample configuration. son-Ruden, 1997). These adhesions repre- These data suggest that the degradation of sent a significant complication if a second poly=4HB) in vivo depends in part on surface operation is necessary, thereby increasing area, and that the mechanical properties of the risk of rupturing the heart or a major poly=4HB) implants are likely to undergo a vessel, and prolonging the overall duration gradual change rather than the more abrupt of the operation. In an initial study, native changes seen with other synthetic absorb- pericardium was excised from 18 sheep, and 14 20 Applications of PHAs in Medicine and Pharmacy

replaced with a nonwoven poly=3HB) patch but with their pericardium left open. It was =Malm et al., 1992a). Patches were then noted however that there might be a species harvested between 2 and 30 months after variation, and also that the duration of the the operation, examined for adhesions, studies was different. Also, in comparison infection, and inflammatory response, and with Malm's sheep study, the calves in this compared with controls where native peri- study had been subjected to bypass, which cardium had been removed and left open. was considered more clinically relevant. Moderate adhesions were present in all the controls, whereas no adhesions developed in 7.1.2 14 of the animals receiving poly=3HB) Artery Augmentation patches. Interestingly, the pericardium was Non-woven patches of poly=3HB) have been regenerated in all animals receiving a patch, evaluated in the augmentation of the pul- with the surface of the patches being monary artery as scaffolds for the regener- completely covered with mesothelium-like ation of arterial tissue in low-pressure cells after two months, and a dense under- systems =Malm et al., 1994). A total of 19 lying collagen layer developing over 12 lambs was used for the trial, with 13 months. A pronounced tissue response to receiving poly=3HB) patches, and six receiv- the patch was observed, with the polymer ing Dacron patches as a control group. No being slowly phagocytosed by polynuclear aneurysms were observed in either group, macrophages ± a finding which has led and the pores of the non-woven poly=3HB) others to question the biocompatibility of the were small enough to prevent bleeding. All patch =Tomizawa et al., 1994). Polymer the patches were harvested between 3 and 24 remnants were still present after 24 months, months, and endothelial layers were found and some macrophages were still found at 30 on both patch materials. Beneath the endo- months, but no platelet aggregates were thelium-like surface, the configuration detected. closely resembled native artery, with smooth Following animal studies with the po- muscle cells, collagen and elastic fibers in ly=3HB) patch, a randomized clinical study the poly=3HB) explants. By contrast, a thin of 50 human patients admitted for bypass collagenous layer had formed under the surgery and/or valvular replacement was endothelium lining of the Dacron implants undertaken =Duvernoy et al., 1995). Using due to the well-known inflammatory reac- computed tomography =CT), 39 of these tion to Dacron fiber, and a dense infiltration patients =19 with the patch and 20 without) of lymphocytes was present. As in the case of were examined for the presence of adhesions the pericardial studies, the nonwoven po- at 6and 24 months, and a lower incidence of ly=3HB) patch was phagocytosed by poly- postoperative adhesions was reported for the nucleated macrophages, and macrophages group receiving PHA patches, based on the persisted, even at 24 months. However, no presence of fat located between the patch and platelet aggregates were found on the lumi- the cardiac surface. nal surface. In contrast to these studies, Nkere et al Highly porous foam patches of poly=4HB) =1998) found no significant difference in a seeded with endothelial, smooth muscle short-term study of adhesion formation cells, and fibroblasts have also been eval- among calves undergoing bypass surgery uated in artery augmentation, and with good with and without the poly=3HB) patch, as results =Stock et al., 2000a). A total of six cell- well as calves not undergoing bypass surgery seeded patches, and one unseeded control 7 Applications 15 patch were implanted into the pulmonary Attention is beginning to focus on the use artery of sheep. Echocardiography and ex- of PHAs in absorbable stents as well as amination of the cell-seeded explants at 4, 7 coatings, often in combination with drug and 20 weeks indicated that progressive delivery systems. Van der Giessen et al. tissue regeneration had occurred, but with =1996) evaluated several bioabsorbable poly- no evidence of thrombus, dilation, or steno- mers, including poly=3HB-co-3HV), as can- sis. Examination of the control patch at 20 didate biomaterials for cardiovascular stents. weeks revealed slight bulging at the site of Strips of polymer were deployed on the implantation, and less tissue regeneration. surface of coil wire stents, and implanted in porcine coronary arteries of 2.5 ± 3.0 mm diameter. After four weeks, most of the 7.1.3 materials tested, including poly=3HB-co- Atrial Septal Defect Repair 3HV), had provoked extensive inflammatory Malm et al. =1992c) also tested the efficacy of responses and fibrocellular proliferation. the nonwoven poly=3HB) patch in the repair However, these results were shown to be of atrial septal defects created in six calves. inconsistent with in vitro results, and that Implants were evaluated between 3 and 12 other factors such as implant geometry, months, and complete endothelial layers implant design, and degradation products facing the right and left atrium were ob- may have been responsible for some of the served, with a subendothelial layer of colla- observed response. It was also noted that the gen and some smooth-muscle cells. As polymers were not sterilized prior to im- before, the patch was degraded by polynu- plantation. clear macrophages, with small particles of The homopolymer, poly=3HB), has also polymer still present at 12 months, and a been fabricated into a cardiovascular stent foreign body reaction persisting. Nonethe- =Schmitz and Behrend, 1997), and tested in a less, the patches prompted the formation of rabbit model =Unverdorben et al., 1998). It regenerated tissue that resembled native was also reported that poly=3HB) stents atrial septal wall, and had sufficient strength plasticized with triethyl citrate =Behrend to prevent the development of shunts in the et al., 2000b) and fabricated by laser cutting atrial septal position. of a molded construct had an average elastic recoil of about 20 ± 24% immediately after 7.1.4 dilation, and of 27 ± 29% after 120 h in vitro Cardiovascular Stents =Behrend et al., 1998). After implantation One of the main problems with the use of into the arteries of rabbits, the poly=3HB) metallic stents in cardiovascular applications stents instigated a temporary intimal prolif- is the subsequent restenosis that can result eration, and were observed to degrade fairly from excessive growth of the blood vessel rapidly in vivo. wall. This is believed to be due =at least in part) to irritation caused by the metallic stent 7.1.5 on the vessel wall. A potential solution to this Vascular Grafts problem may lie in the development of an Vascular grafts are currently inserted to absorbable stent that can prevent reocclu- repair or replace compromised blood vessels sion of the vessel in the short term, but then in arterial or venous systems that have been be absorbed so that it does not cause any subjected to damage or disease, for example persistent irritation of the vessel wall. atherosclerosis, aneurysmal disease or trau- 16 20 Applications of PHAs in Medicine and Pharmacy

matic injury. For the larger-diameter vessels, sections of carotid artery harvested from synthetic grafts are frequently employed, lambs. After seven days, the seeded conduits and these can be impregnated with protein were used to replace 3 ± 4-cm abdominal to make them completely impervious to aortic segments in lambs. The lambs were blood, and thereby ready for anastomotic sacrificed at 10 days and 5 months after procedures. Several studies have shown, surgery, and the conduits compared with however, that when protein substrates are unseeded controls. All control conduits impregnated into grafts they may promote became occluded during the study, but the undesirable immunological reactions. In cell-seeded tissue-engineered grafts re- order to try and develop an improved sealant mained patent =open to blood flow), except for a synthetic graft, Noisshiki and Komat- for one stricture, and no aneurysms were suzaki =1995) investigated the possible use of observed. This contrasts sharply with results poly=3HB-co-4HB) as a graft coating, the obtained using a polyglactin-PGA composite coated grafts being implanted into dogs and that was limited by high porosity, stiffness, examined at 2 and 10 weeks. Subsequently, it and a relatively short degradation time, and was noted that degradation of the polymer developed aneurysms within a few weeks. had already started after two weeks. Histologic analysis of the poly=3HO-co- Marois et al. =1999c, 2000) investigated the 3HH)-PGA grafts was reported to reveal an use of poly=3HO-co-3HH) as an impregna- insignificant inflammatory response, with tion substrate. Polyester grafts impregnated increased cell density, collagen formation with poly=3HO-co-3HH) were implanted in and mechanical properties that were ap- rats, and compared with both protein- and proaching those of native aorta. fluoropolymer-impregnated grafts for peri- It has also been proposed that poly=3HB) ods ranging from 2 to 180 days. No infiltra- might be used to repair severed blood vessels tion of tissue into the poly=3HO-co-3HH)- by the insertion of a tube of this material impregnated graft occurred because of the =Baptist and Ziegler, 1965). presence of the polymer; moreover, polymer degradation was found to be very slow with 7.1.6 just a 30% reduction in molecular weight Heart Valves after six months. Tissue response, after an Perhaps the most remarkable results with initial acute phase seen in all grafts, was PHA polymers have been obtained in the reported as generally mild, and additional development of cell-seeded tissue-engi- investigations with this biomaterial were neered heart valves. These valves promise recommended. to provide unique solutions to the deficien- The elastomeric polymer, poly=3HO-co- cies of mechanical and animal valves cur- 3HH), has also been evaluated as a compo- rently in clinical use, such as the need for nent of an autologous cell-seeded tissue- anticoagulant therapy and repeat surgery to engineered vascular graft in lambs =Shum- replace defective or even outgrown valves in Tim et al., 1999). Tubular conduits =7 mm young children. In initial studies in this area, diameter) comprising a nonwoven PGA scaffolds seeded with autologous vascular mesh on the inside and layers of po- cells and based on porous PGA and PLA had ly=3HO-co-3HH) outside were prepared, been used to replace a single pulmonary and seeded with a mixed cell population of valve leaflet in lambs, but attempts to replace endothelial cells, smooth muscle cells, and all three pulmonary valve leaflets had failed fibroblasts, obtained by the expansion of due to the relatively high stiffness and 7 Applications 17 rigidity of the PGA-PLA biomaterials. How- valve. Two components contributed to this ever, when the leaflets were replaced with success. First, the use of poly=4HB), which porous poly=3HO-co-3HH), and sutured to a was coated on a PGA mesh to provide a conduit composed of a poly=3HO-co-3HH) rapidly degrading yet flexible scaffold; and film sandwiched between layers of PGA second, the use of an in vitro pulse duplicator mesh, these difficulties were overcome system. After 20 weeks in vivo, the mechan- =Stock et al., 2000b). Echocardiography of ical properties of the valve were reported to the seeded constructs implanted in lambs resemble those of the native valve, and indicated no thrombus formation with only histologic analysis showed uniform, layered mild, nonprogressive, valvular regurgitation cuspal tissue with endothelium =see Figure 8 up to 24 weeks after implantation. Histologic of Schoen and Levy, 1999). Echocardiogra- examination revealed organized and viable phy demonstrated mobile, functioning leaf- tissue, without thrombus formation. Nota- lets without stenosis, thrombus, or aneur- bly, thrombus developed on all leaflets in the ysm to 20 weeks, and importantly, the inner unseeded control scaffolds after four weeks. diameter of the valve construct was found to After six weeks in vivo, no PGA remained, have increased from 19 mm at implant to but poly=3HO-co-3HH) was still substantial- 23 mm at 20 weeks. The latter finding is ly present with a slight reduction in molec- particularly exciting for the development of a ular weight =26%). A variant of this scaffold, tissue-engineered heart valve that can be with leaflets derived from poly=3HO-co- used in young children, and will grow with 3HH) sandwiched between PGA mesh, the child. has also been evaluated under pulsatile flow in vitro =Sodian et al., 1999). Under these 7.2 conditions, it was shown that vascular cells Dental and Maxillofacial attached to the scaffold, proliferated, and oriented in the direction of flow after four 7.2.1 days. Guided Tissue Regeneration The design of the heart valve scaffolds have In guided tissue regeneration, barrier mem- been further refined in subsequent studies, branes are used to encourage regeneration of and other PHA polymers have also been new periodontal ligament by creating a space evaluated =Sodian et al., 2000a). Sodian et al. or pocket that excludes gingival connective =2000b,c,d) also described the fabrication of a tissue from the healing periodontal wound, functional tissue-engineered heart valve en- and also by preventing the downgrowth of tirely from porous poly=3HO-co-3HH), and epithelial tissue into the wound. Galgut et al. seeding of the scaffold with vascular cells =1991) evaluated the histologic response of from ovine carotid artery which resulted in rats to poly=3HB-co-3HV) membranes that cellular in-growth into the pores and forma- could be used in this application, and found tion of a confluent layer under pulsatile flow that the membranes were well tolerated. conditions. Compared with Gore-TexTM =polytetrafluoro- Recently, in one of the most astonishing ethylene, PTFE) membranes, little down- results of tissue engineering described to growth of epithelial tissue was observed with date, Hoerstrup et al. =2000) succeeded in poly=3HB-co-3HV). developing a PHA-based heart valve scaffold During the closure of palatal defects, in lambs that was completely replaced at mucoperiosteal flaps are frequently moved eight weeks by a functional trileaflet heart to the midline of the palate, leaving two areas 18 20 Applications of PHAs in Medicine and Pharmacy

of denuded bone adjacent to the dentition. poly=3HB-co-3HV) membranes successfully These wounds heal by migration of kerati- to increase the height of the rat mandible. In nocytes and fibroblasts, as well as by wound two-thirds of the rats, the space created by contraction. Later, the formation of scar the poly=3HB-co-3HV) membrane was com- tissue occurs, and it is believed that attach- pletely filled with bone by six months, ment of this tissue to areas of the denuded although in some cases soft tissue migrated bone can disturb subsequent maxillary through ruptures in the membranes, there- growth, or might affect the development of by inhibiting bone formation. In contrast, dentition. Using beagle dogs, Leenstra et al. bone formation was negligible when mem- =1995) investigated the use of nonporous branes were not used. It was noted that while poly=3HB-co-3HV) films to keep the muco- this biomaterial might form the basis of a periosteum and bone separated until the barrier membrane, some modifications to transition of teeth was completed =at about the physical properties would be required. 24 weeks). At two weeks, it was reported that Barrier membranes of poly=3HB-co-3HV) one of the poly=3HB-co-3HV) films had have been reinforced with polyglactin fibers deformed; however, this was attributed to and used to cover dental implants placed in the method of insertion. At 8 and 12 weeks, fresh extraction sockets in dogs =Gotfredsen the films were unimpaired and surrounded et al., 1994). However, very poor results were by fibrous capsules, and it was concluded obtained. After 12 weeks, inflammatory that poly=3HB-co-3HV) films were more infiltrates were seen adjacent to the po- suitable for this procedure in terms of ly=3HB-co-3HV)-polyglactin membrane that mechanical properties and tissue response interfered with bone healing, and less bone than was PLA or PCL. fill was observed compared with control sites with no membrane. Given the results of 7.2.2 Kostopoulos and Karring =1994a,b), and the Guided Bone Regeneration known inflammatory response of tissues to In addition to using barrier membranes to PGA degradation products, the observed create new periodontal ligament, mem- result might be attributed to the polyglactin branes can also be used to generate new component of the membrane. bone in jaw bone defects, as well as to increase the width and height of the alveolar 7.3 ridge. Kostopoulos and Karring =1994a) Drug Delivery reported successful bone regeneration in jaw bone defects in rats using poly=3HB-co- The potential use of poly=3HB) and po- 3HV) membranes to create spaces for bone ly=3HB-co-3HV) in drug delivery has been fill. Mandibular defects were produced in evaluated in a number of studies, and the rats and either covered with poly=3HB-co- field has been reviewed several times =Hol- 3HV) membranes, or left uncovered. During land et al., 1986; Juni and Nakano, 1987; the following 15 to 180 days, increasing bone Koosha et al., 1989; Pouton and Akhtar, fill was achieved with poly=3HB-co-3HV) 1996; Nobes et al., 1998, Scholz, 2000). membranes, whereas in the uncovered con- Studies have included investigations of these trol group ingrowth of other tissues oc- polymers as subcutaneous implants, com- curred, and only 35 ± 40% of the defect area pressed tablets for oral administration, and was filled with bone after 3 ± 6months. microparticulate carriers for intravenous Kostopoulos and Karring =1994b) also used use. 7 Applications 19

7.3.1 was decreased, leading to reduced compres- Implants and Tablets sibility, and more rapid influx of fluid into In the early 1980s, Korsatko et al. =1983a,b) the tablet. The higher molecular-weight studied the release of a model drug, 7- dextrans also released more rapidly than hydroxyethyltheophylline =HET), from po- fluorescein, but this was attributed to the ly=3HB) tablets prepared by homogeneously creation of more porous and hydrophilic compounding and compressing the polymer matrices and led to the testing of porosigen and the drug. In both in vitro and in vivo =pore-forming) additives as a means of experiments, the drug was released in a controlling the rate of drug release. Two linear manner, but the rate of release from additives, microcrystalline cellulose and lac- subcutaneous implants in mice was found to tose, were tested, and both were found to be about two- to three-fold slower than in enhance the rate of drug release from the vitro. At a drug loading of 10%, sustained modified tablets. release was observed for approximately 10 Release profiles of 5-fluorouracil =5-FU) weeks in vitro, and up to 20 weeks in vivo. At from melt-pressed poly=3HB) disks have loadings up to 30%, release could be sus- been examined in vitro over a range of drug tained for up to 50 days; however, at concentrations =10 ± 50%) by Juni and Naka- substantially higher levels =60 ± 80%) all the no =1987). The rate of release was found to drug was released within 24 h. Changes in increase with drug loading, and complete tablet compaction pressures did not alter the release was observed in five days at a 50% release rates. In subsequent studies, Korsat- loading. Gangrade and Price =1992) also ko et al. =1987) evaluated the influence of used melt compression to prepare PHA drug poly=3HB) molecular weight on drug re- delivery systems with lower porosity, but lease, and found the release of the anti- chose the lower-melting poly=3HB-co-3HV) hypertensive drug midodrin-HCl from com- copolymer, and incorporated progesterone at pressed tablets to be increased as the loadings of 5 to 50%. Consistent with the polymer's molecular weight increased from findings of others, increased loadings re- 3000 to 600,000. In comparison, when po- sulted in faster rates of release, with 85% of ly=3HB)-coated granules of the beta-blocker, the drug being released in three days at a Celiprolol-HCl were prepared using a fluid 50% loading compared with 50% at a 5% bed dryer system, it was found that smaller loading. amounts of the higher molecular-weight The homopolymer, poly=3HB), has been polymers were required to slow the release evaluated as a potential candidate for the of Celiprolol-HCl, presumably because of development of a gastric retention drug improved film formation at higher molec- delivery device that could extend the absorp- ular weight. tion period of a drug from the stomach Gould et al. =1987) investigated the release =Cargill et al., 1989). However, further devel- of fluorescein, and dextrans labeled with opment was not pursued because no degra- fluorescein, from tablets of poly=3HB-co- dation of the device was observed in a 12-h 3HV) prepared by direct compression. Con- period during an in vitro dissolution assay. sistent with the observations of Korsatko Jones et al. =1994) developed an auricular et al. =1983a,b), the rate of release was found poly=3HB-co-3HV) implant for cattle con- to increase significantly at higher loadings. taining metoclopramide at a loading of 50% Faster rates of release were also observed as as a prophylactic treatment for fescue tox- the percentage of valerate in the copolymer icosis =which is caused by cattle grazing on 20 20 Applications of PHAs in Medicine and Pharmacy

endophyte-infected fescue). The implant poly=3HB-co-3HV) copolymers, contribu- was prepared by melt compression, and ting to a slower release of the drug from released metoclopramide at an effective rate the homopolymer in vitro. of 12 mg per day in vivo. Hasirci et al. =1998) described the develop- Compression-molded compacts of po- ment of poly=3HB-co-3HV) rods containing ly=3HB) loaded with tetracycline have been the antibiotic Sulperazone for the treatment evaluated in the treatment of periodontal of osteomyelitis. Rods loaded with 20 and disease =Collins et al., 1989; Deasy et al., 50% of the drug were prepared by adding 1989). Using in vitro studies, poly=3HB) granules of the antibiotic to poly=3HB-co- compacts loaded with 50% tetracycline were 3HV) solvent solutions, and molding the developed that could deliver therapeutic resulting pastes into rods. These rods were levels of the antibiotic for eight to nine days. then introduced into rabbit tibias containing Six patients with gingivitis were treated with metal implants infected with Staphylococcus these compacts, and their saliva was moni- aureus =obtained from chronic osteomyelitis tored for the release of tetracycline. Thera- patients). After 15 days the infection had peutic levels of tetracycline were detected been eradicated. A similar approach to the over the 10-day study period, and an im- treatment of osteomyelitis using poly=3HB- provement in the gingival condition from co-3HV) rods containing sulbactam-cefoper- moderate to mild inflammation was report- azone has also been reported =Yagmurlu ed. However, when the treatment was stop- et al., 1999). Recently, Korkusuz et al. =2001) ped the improvement was not maintained. evaluated the use of poly=3HB-co-4HB) and Subcutaneous implants of poly=3HB) con- poly=3HB-co-3HV) rods as antibiotic carriers taining gonadotropin-releasing hormone for the treatment of osteomyelitis. A bone =GnRH) have been tested for their ability infection, experimentally induced with to release this hormone, and stimulate S. aureus, was effectively treated with sol- luteinizing hormone =LH) secretion, pro- vent-blended rods containing Sulperazone mote preovulatory follicle growth, and in- or Duocid. It was noted that the poly=3HB-co- duce ovulation in acylic sheep =McLeod 4HB) rods were preferred as they were less et al., 1988). In comparison with oil-based rigid and easier to handle than the poly=3HB- formulations, the poly=3HB) implants con- co-3HV) rods. taining 40 ± 50 mg of GnRH consistently Kharenko and Iordanskii =1999) prepared produced elevated plasma levels of LH for poly=3HB) tablets containing the vasodilator, periods of two to four days, and a high diltiazem, with drug loadings up to about incidence of ovulation was obtained, partic- 45%, and monitored release rates of these ularly when two implants per animal were delivery systems in vitro. Near-complete used. release of the drug was observed at the Akhtar et al. =1989, 1991, 1992) investigat- highest loading concentrations, with slower ed the release of a model drug, methyl red, release being observed at lower loadings. from both solution-cast and melt-processed films of poly=3HB) and poly=3HB-co-3HV), 7.3.2 and examined the effect of varying the Microparticulate Carriers temperature during polymer crystallization. A number of groups have examined the It was found that the faster-crystallizing potential use of poly=3HB) and poly=3HB-co- homopolymer, poly=3HB), was better able to 3HV) polymers as microparticulate carriers trap methyl red than the slower-crystallizing for drug delivery. In general, these systems 7 Applications 21 have been produced using solvent evapora- ly=3HB) microspheres were found to be tion techniques, and variables such as drug somewhat irregular in shape, which was loading, polymer composition, molecular attributed to the highly crystalline nature of weight, crystallization rate, particle size, the homopolymer. and the use of additives have been inves- Brophy and Deasy =1986) examined the tigated. Although there are exceptions, the release profiles of sulphamethizole from following observations are fairly typical: =1) poly=3HB) and poly=3HB-co-3HV) micro- Increased valerate content in copolymers of particles =53 ± 2000 mm) formed by grinding poly=3HB-co-3HV) usually slows the rate of a solvent-evaporated matrix of these compo- drug release, presumably because the co- nents, and reported that increased rates of polymers are less crystalline than po- release were observed when the molecular ly=3HB). Incorporation of valerate into po- weight of poly=3HB) was increased. This ly=3HB) also tends to yield microparticles observation was attributed to poor distribu- that are less susceptible to physical damage tion of the drug in the highly crystalline than poly=3HB); =2) Smaller particle sizes poly=3HB) polymer. As anticipated, faster decrease loading capacity, but increase the rates of release were also observed as the rate of drug release; =3) Small changes in poly=3HB) particle size was decreased, and polymer molecular weight have little impact as drug loading was increased. Incorpora- on release rates; however, large changes can tion of valerate into the poly=3HB) polymer increase crystallinity and lead to enhanced chain decreased the release rate, presumably release rates; =4) Drug release from po- on account of the improved distribution of ly=3HB) and poly=3HB-co-3HV) is complet- the drug. Overcoating of the poly=3HB) ed well before any significant degradation of microparticles with PLA reduced the burst the polymers has begun, so drug release is effect =the initial, rapid release of the drug entirely diffusion controlled; and =5) Lower from the delivery device surface), as well as drug loadings reduce the release rate. significantly reducing the overall release One of the earliest studies of poly=3HB) rate. A sustained release of sulphamethizole microspheres in drug delivery was carried was demonstrated in vivo when poly=3HB) out by Bissery et al. =1983, 1984a). Using a microparticles =425 ± 600 mm) loaded at 50% solvent evaporation technique, 14C-labeled were administered intravenously to dogs. poly=3HB) microspheres =1 ± 12 mm) were Release was complete in about 24 h, and prepared and found, as expected, to concen- correlated well with in vitro results. trate primarily in the lungs of mice upon Slower release of several anticancer anti- intravenous administration. However, when biotics, including doxorubicin, aclarubicin, the microspheres were loaded with an anti- 4'-O-tetrahydropyranyl doxorubicin, bleomy- cancer agent, lomustine [N-=2-chloroethyl)- cin, and prodrugs of 5-fluoro-2'-deoxyuri- N'-cyclohexyl-N-nitrosourea; CCNU], and dine from poly=3HB) microspheres has been administered to Lewis lung carcinoma-bear- reported =Juni et al., 1985, 1986; Juni and ing mice, little effect was observed =Bissery Nakano, 1987; Kawaguchi et al., 1992). For et al., 1985). From in vitro studies it was example, at a 13% loading of aclarubicin- found that the drug was completely released HCl, poly=3HB) microspheres with a mean from the microspheres in 24 h at a loading of diameter of 170 mm were reported to release 7.4%, compared with a release time of over only 10% of the drug over five days in vitro. 90 h when PLA microspheres were used The observed release rates could, however, be =Bissery et al., 1984b). Notably, the po- increased by the incorporating fatty acid 22 20 Applications of PHAs in Medicine and Pharmacy

esters into the poly=3HB) microspheres. particles with the smallest sizes. Linhardt These esters were believed to facilitate drug et al. =1990) also considered using po- release by forming channels in the po- ly=3HB) and poly=3HB-co-3HV) as vaccine ly=3HB) matrix =Kubota et al., 1988). Abe delivery vehicles. et al. =1992a,b) also reported faster release of The effect of several different parameters an anticancer agent, lastet, from poly=3HB) on the release of progesterone =a low microspheres when acylglycerols were in- molecular-weight model drug) from po- corporated into the microspheres. ly=3HB) and poly=3HB-co-3HV) micro- In vivo, poly=3HB) microspheres contain- spheres prepared with an emulsion solvent ing two prodrugs of 5-fluoro-2'-deoxyuridine evaporation technique has been evaluated by were reported to induce higher antitumor Gangrade and Price =1991). Using scanning effects against P388 leukemia in mice when electron microscopy =SEM), the use of compared with administration of the free gelatin as an emulsifier was shown to prodrugs over five consecutive days =Kawa- provide more spherical microspheres than guchi et al., 1992). when using polyvinyl alcohol, sodium lauryl Koosha et al. =1987, 1988, 1989) reported sulfate or methyl cellulose, and that smooth- the use of high-pressure homogenization to er surfaces were obtained when the solvent produce poly=3HB) nanoparticles contain- was switched from chloroform to methylene ing prednisolone. At drug loadings up to chloride. Generally, poly=3HB) micro- 50%, a biphasic release pattern was observed spheres had very rough surfaces which in vitro, with an initial burst effect followed became smoother as valerate was incorpo- by a slow release of the drug that was rated. Interestingly, release of the drug from complete in one to two days. Similar results poly=3HB-co-9%3HV) was slower than from were observed with tetracaine. Akhtar et al. either poly=3HB) or poly=3HB-co-24%3HV). =1989) has also prepared smaller poly=3HB) Examination of the internal surfaces of these particles =20 ± 40 mm) by spray-drying, and microspheres by SEM revealed fewer cavities reported fairly rapid release of a model drug and less porosity for the poly=3HB-co- =methyl red) from this matrix. 9%3HV) microspheres, and this was con- Microspheres of poly=3HB) have been sistent with the slower release observed. evaluated in vivo as controlled release sys- Porosity was also found to decrease when the tems for the oral delivery of vaccines that microspheres were prepared at increasing could potentially protect vaccine antigens temperatures between 25 and 40 8C. Typi- from digestion in the gut, and target delivery cally, 60 ± 100% of the drug was released to Peyer's patches =Eldridge et al., 1990). from these microspheres in 12 h at drug When a single oral dose of poly=3HB) loadings up to 12%. microspheres containing coumarin was ad- Embleton and Tighe =1992a,b) also inves- ministered to mice, very good absorption of tigated the effects of increasing the valerate microspheres =of diameter <10 mm) was content of poly=3HB-co-3HV), temperature, observed in the Peyer's patches 48 h later. and molecular weight on microsphere for- Conway et al. =1996, 1997) have evaluated mation, and obtained results consistent with the adjuvant properties of poly=3HB) micro- those of Gangrade and Price =1991). When spheres in vivo, and observed a potent 10 ± 50% PCL was incorporated into these antibody response to encapsulated bovine poly=3HB-co-3HV) microspheres, a system- serum albumin =BSA), with the highest titer atic increase in porosity was observed with being generated to preparations containing increasing PCL content, that was attributed 7 Applications 23 to the elution of PCL from the hardened Depending upon valerate content, between microcapsule wall once the poly=3HB-co- 42 and 90% of the tetracycline was released 3HV) had precipitated during formation at 100 h, with loadings up to about 11%. =Embleton and Tighe, 1993). Porosity was Interestingly, it was found that molecular also found to increase significantly when weight variation in the poly=3HB-co-3HV) polyphosphate-Ca2‡ complexes were intro- compositions tested might have influenced duced into poly=3HB-co-3HV) microspheres the observed release of tetracycline. Encap- =Gürsel and Hasirci, 1995). sulation efficiency of the tetracycline hydro- Atkins and Peacock =1996b) also used PCL chloride salt was significantly less than the to prepare microcapsules =21 ± 200 mm) from neutral form of the antibiotic. a blend of poly=3HB-co-3HV)/PCL=20%) A novel approach for preparing PHA drug with an inner reservoir of BSA loaded in delivery systems =Nobes et al., 1998) involves an agarose core. The protein encapsulation encapsulating a given drug during the in was, however, low =<12 %) and only slightly vitro enzymatic formation of poly=3HB) influenced by loading. Release from the granules =Gerngross and Martin, 1995). microcapsules loaded with up to 50% pro- Using this technique, it was possible to tein was observed in vivo for about 24 days. encapsulate approximately 4.5% of a model Microspheres of poly=3HB) have been drug compound, Netilmicin, compared with evaluated as potential embolization agents between 4.1 and 17% using solvent evapo- in renal arteries =Kassab et al., 1999), and ration. with rifampicin as a chemoembolization Wang and Lehmann =1999) recently pre- agent =Kassab et al., 1997). Release of rifam- pared poly=3HB) microspheres containing picin during in vitro studies was consistent levonorgestrel with an average particle size with other controlled release studies, with of 64 mm, and found that this system pro- near-complete release of the drug being longs the release of the drug by 1.8-fold observed at high loadings =40%) in 24 h, compared with administration of the drug and a somewhat prolonged release at lower alone. The microspheres were also found to loadings. Renal angiograms obtained before be effective in vivo, inducing a contraceptive and after embolization with poly=3HB) effect in mice. Chen et al. =2000) recently microspheres =120 ± 200 mm) using a con- described microspheres =30 ± 40 mm) loaded trast agent showed that 10 mg of the micro- with diazepam, and reported the character- spheres was sufficient to slow renal arterial istic biphasic release pattern with an initial blood flow, with subsequent partial occlu- burst effect. sion of the pre-capillaries in two adult dogs. Andersson et al. =1999) disclosed the use When a second injection was given, com- of supercritical fluid technology to incorpo- plete embolization was achieved. Histo- rate the water-insoluble Helicobacter pylori pathologic examination of the kidneys re- adhesion protein A =HpaA) into poly=3HB) vealed changes consistent with renal artery particles. This was achieved by preparing an obstruction and blockade of the blood supply emulsion with the protein, polymer, and to the kidneys. methylene chloride, and then extracting the In addition to using poly=3HB) compacts, organic solvent from the emulsion with microspheres of poly=3HB-co-3HV) have supercritical carbon dioxide to induce par- been used to deliver tetracycline and its ticle formation. Particles containing 0.6% hydrochloride salt for the treatment of protein were produced, with sizes of 1 ± periodontitis =Sendil et al., 1998, 1999). 3 mm. 24 20 Applications of PHAs in Medicine and Pharmacy

7.4 material was used to bridge an irreducible Prodrugs gap of 10 mm in rat sciatic nerve, and the results were compared to an autologous In 1999, it was discovered that poly=4HB) nerve graft =Hazari et al., 1999b). Good could be used as a prodrug of 4-hydroxy- axonal regeneration in the poly=3HB) con- butyrate =Williams and Martin, 2001). In duits with a low level of inflammatory this study, rats were dosed =by gavage) with infiltration was observed over 30 days, low molecular-weight polymers of po- although the rate and amount of regener- ly=4HB) =138 mg kgÀ1), and the serum was ation in the poly=3HB) conduit did not fully assayed for the presence of monomer. The match that of the nerve graft. serum concentration of 4-HB increased to ~ 86 mM within 30 min, and remained ele- 7.6 vated at approximately three- to five-fold the Nutritional Uses baseline value =9 mM) for about 8 h. In contrast, administration of the monomer 7.6.1 resulted in a characteristic rapid increase to Human Nutrition 182 mM within 30 min, followed by a rapid Several groups have evaluated oligomeric decrease to baseline within 2 h. The pro- forms of the ketone body, R-3-hydroxybuta- longed release of the monomer from po- noic acid, as an alternative to the sodium salt ly=4HB) might potentially be beneficial in of the monomer, for potential nutritional the treatment of narcolepsy, alcohol with- and therapeutic uses. Use of these polymeric drawal, and several other indications. The forms might provide controlled release therapeutic potential of poly=4HB) was also systems for the monomer and, importantly, alluded to recently =Sudesh et al., 2000). overcome the problems associated with administering large amounts of sodium 7.5 ion in vivo. Tasaki et al. =1998) reported the Nerve Repair results of infusing dimers and trimers of R- 3-hydroxybutyrate into rats, as well as ex- Partly on account of the piezoelectric proper- posure of these compounds to human serum ties of poly=3HB), interest has arisen in the samples and liver homogenate. Although use of this polymer for nerve repair mixtures of these compounds were not =Aebischer et al., 1988). The basic approach hydrolyzed by human serum, the monomer involves aligning severed nerve ends within was liberated upon exposure to the liver a small tube of poly=3HB), thus avoiding the homogenate as well as after infusion in rats. need for sutures. Hazari et al. =1999a) and In vitro, the monomer was also liberated Ljungberg et al. =1999) evaluated the use of a after incubation with the enzyme carboxyl- nonwoven poly=3HB) sheet as a wrap to esterase. repair transected superficial radial nerves in Veech =1998, 2000) and Martin et al. =2000) cats for up to 12 months. Axonal regener- have evaluated oligomers and oligolides of R- ation was shown to be comparable with 3-hydroxybutyrate in vivo, and observed closure with an epineural suture for a nerve release of the ketone body over prolonged gap of 2 ± 3 mm, and that the inflammatory periods. Potential uses of these delivery response created by poly=3HB) was also systems might include seizure control, re- similar to that found in primary epineural duction of protein catabolism, appetite sup- repair. In a subsequent study, the same pression, control of metabolic disease, use in 7 Applications 25 parenteral nutrition, increased cardiac effi- reinforced Vicryl. After 12 weeks, better ciency, treatment of diabetes and insulin- results were obtained with the reinforced resistant states, control of damage to brain poly=3HB) plates relative to the Vicryl plates, cells in conditions such as Alzheimer's, and with the latter frequently leading to nonun- treatment of neurodegenerative disorders ion of the osteotomies, breakage, and angu- and epilepsy. lation. Doyle et al. =1991) reported that poly=3HB) 7.6.2 can be reinforced with hydroxyapatite =HA) Animal Nutrition to increase its stiffness to a level approaching The potential use of poly=3HB) and po- that of cortical bone =7 ± 25 GPa). At a HA ly=3HB-co-3HV) polymers as a source of loading of 40% wt., a poly=3HB)/HA com- animal nutrition has been evaluated in vivo. posite had a Young's modulus value of Brune and Niemann =1977a) initially report- 11 GPa, although strength decreased from ed studies in rats, and subsequently de- ~ 40 MPa for poly=3HB) to ~ 20 MPa for the scribed the digestion of poly=3HB) in pigs composite. Under in vitro conditions in =Brune and Niemann, 1977b). In the latter buffered saline at 37 8C it was noted that work, when whole cells containing po- the modulus of the filled poly=3HB) sample ly=3HB) were used as a nutrient source, decreased more rapidly, falling from 9 GPa approximately 65% of the poly=3HB) was to 4 GPa over four months at a loading of excreted. Forni et al. =1999a,b) found the 20% wt. HA. Bending strength of the same digestibility of poly=3HB-co-3HV) treated filled sample also fell by about 50% over the with sodium hydroxide to be increased when same time period, from 55 MPa to around compared with the untreated polymer, in 25 MPa. The behavior of poly=3HB) filled both sheep and pigs. Holmes =1988) also with HA was also studied in vivo.No reported that poly=3HB) is degraded in the significant differences between implants bovine rumen, while Peoples et al. =1999) derived from poly=3HB) or poly=3HB) filled studied the digestion of poly=3HB) and with HA were observed when rivets were poly=3HO-co-3HH) in broiler chicks, con- prepared from these materials and inserted cluding that the available energy from these into predrilled holes in rabbit femurs. Over polymers lies between that provided by time, increased amounts of new bone were carbohydrates and oils. found on the implant surfaces, and by six months the implants were closely encased in new cortical bone. The overall tissue re- 7.7 sponses were considered favorable, and Orthopedic some indication of osteogenic activity for poly=3HB) was noted. Boeree et al. =1993) Several studies of the use of PHA polymers and Galego et al. =2000) have also studied the for internal fixation have been undertaken. mechanical properties of poly=3HB) and Vainionpää et al. =1986) described the use poly=3HB-co-3HV)/HA composites, and of compression-molded T-plates, prepared concluded that they could serve as alterna- from poly=3HB) reinforced with carbon fiber tives to corticocancellous bone grafts. =7%), to fix osteotomies of the tibial diaphy- Since the homopolymer poly=3HB) is sis in rabbits. The implants were fixed to the piezoelectric, it might help to induce new tibia with absorbable PGA sutures, and local bone formation if used as an implant. compared with implants prepared from The addition of other additives might further 26 20 Applications of PHAs in Medicine and Pharmacy

enhance this property, for example bioactive In addition to composites with hydroxy- glasses that upon dissolution and deposition apatite and glass, Jones et al. =2000) tested at an implant surface may encourage new composites of poly=3HB-co-3HV) with tri- bone formation. In this light, Knowles et al. calcium phosphate filler in subcutaneous =1991) studied the piezoelectric character- and femoral implants. These implants were istics of poly=3HB-co-3HV) composites with compared with composites derived from glass fiber at 20, 30 and 40% wt., and found PLA and tri-calcium phosphate, and found the piezoelectric potential output of these to degrade about four times more slowly in composites to be fairly close to that of bone. vivo. In subsequent studies, these composites In an in vitro study, Rivard et al. =1996) were evaluated in vitro and in vivo =Knowles prepared highly porous foams of poly=3HB- and Hastings, 1993a,b), and have also been co-3HV) and seeded these scaffolds with studied with the inclusion of HA =Knowles chondrocytes and osteoblasts. Maximal cell et al., 1992). During in vitro studies of the densities were achieved after 21 days, with poly=3HB-co-3HV)-glass composites, the cellular diffusion taking place throughout glass component was found to be highly the porous foams. soluble in the polymer, and the observed weight loss was attributed to dissolution of 7.8 the glass from the composites. These studies Urology correlated well with observations in vivo. The poly=3HB-co-3HV)-glass composites were In the mid-1960s it was proposed that implanted subcutaneously, and as nonload- poly=3HB) could be used to repair a ureter bearing femoral implants in rats. Initially, by inserting a short tube of this material relatively high cellular activity was observed =Baptist and Ziegler, 1965). More recently, that was attributed to ions being released Bowald and Johansson =1990) described the from the glass =and causing a soft tissue use of poly=3HB-co-3HV) in the develop- reaction), though this activity decreased with ment of a tube for urethral reconstruction. A time. At four weeks in the femoral implants, solution of the poly=3HB-co-3HV) copoly- cells could be seen entering the surface mer was used to coat thin, knitted tubes of porosity formed by the solubilizing glass, Vicryl, and the tubes were then implanted and over time new bone was seen developing into four dogs to replace the urethra. After on the implant surface =Knowles and Hast- six to nine months, it was claimed that a fully ings, 1993a,b). However, when composites functional urethra tissue had been recon- of poly=3HB-co-3HV)/HA and poly=3HB-co- structed in all animals. 3HV)/HA-glass were implanted in rabbit femurs, and evaluated using a mechanical 7.9 push-out test during the first eight weeks in Wound Management vivo, it was found that the former bonded better than the latter. One explanation for 7.9.1 this observation was attributed to the release Sutures of ions from the poly=3HB-co-3HV)/HA- As early as the mid-1960s it was suggested glass composite inducing a soft tissue that poly=3HB) could be used as an absorb- reaction that inhibited the formation of hard able suture =Baptist and Ziegler, 1965). tissue at the implant surface =Knowles et al., Others have also proposed that poly=3HB- 1992; Knowles, 1993). co-3HV) could be used as a suture coating, 8Future Directions 27 and have coated braided sutures of PGA with cence. After one month, the incisions had solutions of this polymer =Wang and Leh- substantially healed, and no coalescence had mann, 1991). occurred. However, no degradation of the film in vivo was observed at one year. 7.9.2 Dusting Powders 7.9.4 Holmes =1985) has produced powders of Soft Tissue Repair poly=3HB) with small particle sizes, and In early studies of the potential uses of proposed that these can be used as medical poly=3HB) it was proposed that this polymer dusting powders, particularly with surgical could be used in the surgical repair of gloves. hernias =Baptist and Ziegler, 1965). Patches of poly=3HB) with a smooth 7.9.3 surface on one side and a porous surface Dressings on the other have been evaluated as resorb- Webb and Adsetts =1986) described wound able scaffolds for the repair of soft-tissue dressings based on volatile solutions of defects, and specifically for the closure of poly=3HB) and poly=3HB-co-3HV) that could lesions in the gastrointestinal tract =Behrend form thin films over wounds; these would be et al., 1999). Using in vitro cell culture, especially useful for emergency treatments. moderate adhesion of mouse and rat intes- These films could potentially prevent air- tine fibroblasts to the patch material was borne bacterial contamination of the wound, observed. When the patches were sutured but would still be permeable to water vapor. over incisions in the stomachs of rats that As the preferred solvents are chlorinated had been closed with two surgical knots, hydrocarbons, however, these solutions adhesion of the patch to the gastric wall was might present other health hazards both to found to be better than that seen with Vicryl the patient and administrator. Steel and patches, and good ingrowth and tissue Norton-Berry =1986) also described wound regeneration was evident on the porous side dressings based on poly=3HB), their method of the implanted poly=3HB) patch. involving the preparation of nonwoven fi- brous materials of poly=3HB) and PHBV that could be used like swabs, gauze, lint or 8 fleece. Future Directions Davies and Tighe =1995) evaluated the use of poly=3HB) fibers as a potential wound With technology now in place to allow the scaffold that could provide a framework for properties of PHA polymers to be tailored to the laying down of a permanent dermal specific applications, coupled with a signifi- architecture. Using in vitro cell attachment cant increase in the need for new absorbable assays with human epithelial cells, it was biomaterials, this class of polymers currently found that pre-treatment of the poly=3HB) appears to have a bright future in medicine fibers with either base or strong acid im- and pharmacy. Indeed, a wide new range of proved cell attachment and spreading. applications for PHA polymers has recently Ishikawa =1996) described the implanta- been described which includes their use as tion of poly=3HB-co-4HB) films in the suture anchors, meniscus repair devices, abdominal cavity of rats between incisions interference screws, bone plates and bone in the skin and intestine to prevent coales- plating systems, meniscus regeneration de- 28 20 Applications of PHAs in Medicine and Pharmacy

vices, ligament and tendon grafts, spinal 9 fusion cages and bone dowels, bone graft Patents substitutes, surgical mesh and repair patch- es, slings, adhesion prevention barriers, skin Patents cited in studies described in this substitutes, dural substitutes, bulking and chapter are listed in Table 2. filling agents, ureteric and urethral stents, vein valves, ocular cell implants, and hemo- stats =Williams, 2000; Williams et al., 2000).

Tab. 2 Patents referenced in this work.

Patent Number Assignee Inventors) Title Date of Publication

WO 88/06866 Brown Aebischer, P., Piezoelectric nerve September 22, University Valentini, R.F., guidance channels 1988 Research Galletti, P.M. Found. WO 99/52507 Astra Andersson, M.-L., Incorporation of active October 21, Aktiebolag Boissier, C., Juppo, substances in carrier 1999 A.M., Larsson, A. matrixes US 3,225,766 W.R. Grace and Baptist, J.N., Ziegler, Method of making December 28, Co. J.B. absorbable surgical 1965 sutures from poly beta hydroxy acids EP 0 349 505 A2 Astra Meditec Bowald, S.F., A novel surgical material January 3, 1990 AB Johansson, E.G. EP 0 754 467 A1 Astra Bowald S.F., A novel surgical material. January 22, Aktiebolag Johansson-Ruden, G. 1997 US 5,116,868 John Hopkins Chen, C.-H., Chen, Effective ophthalmic May 26, 1992 University S.C. irrigation solution EP 355,453 A2. Kanegafuchi Hiraide, A., Katayama, Use of 3-hydroxybutyric February 28, Kagaku Kogyo M. acid as an energy source 1990 Kabushiki Kaisha GB 2 160 208A. Imperial Holmes, P.A. Sterilised powders of December 18, Chemical poly=3-hydroxybutyrate) 1985 Industries, Plc. US 5,480,394 Terumo Ishikawa, K. Flexible member for use as January 2, 1996 WO93/05824 Kabushiki a medical bag Apr. 1, 1993 Kaisha WO 99/32536Metabolix, Inc. Martin, D.P., Skraly, Polyhydroxyalkanoate July 1, 1999 F.A., Williams, S.F. compositions having controlled degradation rates WO 00/04895 Metabolix, Inc. Martin, D.P., Peoples, Nutritional and February 3, O.P., Williams, S.F. therapeutic uses of 2000 3-hydroxyalkanoate oligomers 9 Patents 29

Tab. 2 cont.)

Patent Number Assignee Inventors) Title Date of Publication

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KeyWords:

Key words: Absorbable, bioabsorbable, biodegradable, biocompatible, biodistribution, biomaterial, cardiovascular, dental, drug delivery, implant, medical device, microsphere, nutrition, orthopedic, prodrug, sterilization, suture, tissue engineering, tissue regeneration, urology, wound dressing, polyhydroxyalkanoate &PHA), poly-R-3-hydroxybutyrate &poly&3HB)), poly-R-3-hydroxybutyrate-co-R-3- hydroxyvalerate &poly&3HB-co-3HV)), poly-R-3-hydroxyoctanoate-co-R-3- hydroxyhexanoate &poly&3HO-co-3HH)), poly-4-hydroxybutyrate &poly&4HB)), poly-R-3-hydroxybutyrate-co-4-hydroxybutyrate &poly&3HB-co-4HB)), Tepha, Metabolix