Trix Consisting of Citric Acid and Collagen

Advance Online Publication

Enhanced Neovascular Formation in a Novel Hydrogel Ma- trix Consisting of Citric Acid and Collagen

Mikiko Nagayoshi, MD,1, 2 Tetsushi Taguchi, PhD,3 Hiroyuki Koyama, MD,1, 2 Tsuyoshi Takato, MD,2 Tetsuro Miyata, MD,1 and Hirokazu Nagawa, MD1

Background: Three-dimensional regenerative tissue with large bulk generally requires blood perfusion through a vascular network to maintain its viability, and one promising approach is induction of neovascular growth from the recipient bed into the tissue. To induce ingrowth of a vascular network, it is necessary to furnish the regenerative tissue with a scaffold structure for neovasculature and a delivery system for an angiogenic growth factor. As such a scaffold structure, the present study created novel hydrogel materials by chemically cross-linking alkali-treated collagen (AlCol) with trisuccinimidyl citrate (TSC). Materials and Methods: Many prototypes, consisting of several concentrations of TSC and AlCol, were implanted into the subfascial space of the rat rectus muscle, and 7 days later, the implanted materials were excised for histological analysis. Cross-sections were stained and neovascular development in the materials was evaluated by measuring vessel density, length and number of joints and branches. Results: Significant ingrowth of vascularized granulation was observed in some materials, which surpassed the angiogenic ability of MatrigelTM. Further, combination with basic fibroblast growth factor (bFGF) significantly increased the vascular formation in these gels. Conclusions: The TSC-AlCol gel functioned as a favorable scaffold for neovascular formation and also as a reservoir for controlled delivery of bFGF.

Key words: neovasucular formation, hydrogel, collagen, citric acid, basic fibroblast growth factor

Introduction reproduce or simulate the function of the original tissue or organ. One crucial reason for the unfavorable status is ecent advances in the fields of tissue engineering insufficient development of technology to construct the Rand stem cell biology have produced practical regen- three-dimensional (3-D) structure of regenerative tis- erative tissues, some of which have been applied in clini- sues.3) If these 3-D technologies could be applied for tis- cal settings for therapeutic purposes.1, 2) However, at pres- sue engineering, it would be possible to produce highly- ent, there are few regenerative products that completely organized regenerative tissues with reasonable bulk, which would assure high performance of function and a 1 Division of Vascular Surgery, Department of Surgery, Graduate long lifetime. In the construction of 3-D regenerative tis- School of Medicine, The University of Tokyo, Tokyo, Japan 2Division of Tissue Engineering, The University of Tokyo Hospi- sue, the most important step might be the formation of a tal, Tokyo, Japan vascular system in the tissue.4) All tissues and organs re- 3 Biomaterials Center, National Institute for Materials Science, quire a supply of oxygen and nutrition and have to elimi- Tsukuba, Ibaraki, Japan nate carbon dioxide and other metabolites. When the re- Received: February 23, 2011; Accepted: April 7, 2011 generative tissue is small enough, these processes can be Corresponding author: Hiroyuki Koyama, MD. Division of Tis- accomplished by passive diffusion of environmental flu- sue Engineering, The University of Tokyo Hospital, 7-3-1 Hon- id, suggesting that blood perfusion is not always neces- go, Bunkyo-ku, Tokyo 113-0033, Japan Tel: +81-3-5800-8653, Fax: +81-3-3811-6822 sary in such a situation. In contrast, 3-D regenerative tis- E-mail: [email protected] sue with larger bulk requires blood perfusion through a

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Nagayoshi M, et al.

Fig. 1 Schematic illustration of chemically cross-linking reaction of citric acid derivative (TSC) with alkali-treated collagen (AlCol).

vascular system to maintain the viability of the whole tis- Table 1 Formulation of synthesized TSC-AlCol gels sue, since the range of transportation by passive diffusion AlCol (w/v %) is limited to 100–200 m at maximum.5) TSC (mM) μ 7.5 10 15 20 30 Several approaches have been proposed to develop a vascular system in regenerative tissues, and one promis- 1 NA NA NA NA NA 3 NA NA + + + ing approach is induction of newly-formed vessels from 6 + + + + + the host vasculature by controlling vasculogenesis, angio- 10 + - - - - genesis or arteriogenesis.4) Delivery of angiogenic growth NA: Gels were completely disappeared. factors or endothelial precursor cells potentially promotes +: Granulation developed into gels these mechanisms of neovascularization, and this strat- –: No granulation developed into gels egy has been applied for the treatment of vascular occlu- TSC, trisuccinimidly citrate sive disease, such as ischemic heart disease and chronic limb ischemia.6–8) However, to induce vessel formation in regenerative tissue, it is also important that the tissue is Materials and Methods suitably furnished with a scaffold structure for newly- formed vessels, since it is essential for vessels in vivo to Synthesis of hydrogel matrix from TSC and AlCol have a surrounding extracellular matrix (ECM) as a nat- (TSC-AlCol gel) ural scaffold.9) The vascular wall must be supported by TSC was prepared as described previously.13) Brief ly, ECM, and vascular cells receive several stimulants and citric acid was dissolved in tetrahydrofuran (THF), and signals via the matrix.9) MatrigelTM, a preparation of then hydroxysuccimide (HOSu) and dicyclohexycarbodi- basement membrane, is a well-known biomaterial that imide (DCC) were added. This mixture was concentrated possibly serves as favorable scaffold for neovasculariza- with rotary evaporation under reduced pressure, and tion, though it is difficult to utilize the material for clini- recrystallized to yield TSC. The obtained TSC was char- cal purposes, because it is extracted from mouse sar- acterized by 1H-NMR (JEOL EX-300) and elemental 10, 11) coma. analysis: 1H-NMR (DMSO-d6) δ = 2.8 ppm (s, 12H, suc- As a safe biomaterial to induce neovascular ingrowth, cinimidyl esters CH2 × 6), 3.4 ppm (s, 4H, CH2 × 2), 7.2 we previously developed physical gel matrix which was ppm (s, 1H, OH). Analysis: Calculated for C18H17N3O13: C, an ion complex of atelocollagen and alkali-treated colla- 44.73; H, 3.55; N, 8.69. Found: C, 44.83; H, 3.45; N, 8.58. gen (AlCol) modified with trisuccinimidyl citrate For the synthesis of TSC-AlCol matrices, AlCol (type (TSC).12) In the present study, we created a chemical gel I, derived from pig skin, Nitta Gelatin Inc., Osaka, Japan) matrix for the same purpose. This hydrogel matrix was was dissolved in dimethylsulfoxide (DMSO) to obtain 7.5, formed by chemically cross-linking AlCol with TSC,13–15) 10, 15, 20 and 30% w/v AlCol solutions. Then, 50 μl TSC and can be combined with a potent angiogenic growth solution at a concentration of 1, 3, 6 or 10 mM was added factor, basic fibroblast growth factor (bFGF). Application to 200 μl of each AlCol diluent. The TSC-AlCol mix- of this biomaterial in regenerative tissue is expected to tures were poured into flat containers so as to be 1 mm lead to development of a vascular network in the regen- thick and incubated for 24 h at 37°C to chemically cross- erative tissue after implantation in vivo. link AlCol with TSC, forming gel sheets (Fig. 1).

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Neovascular Formation in Collagen Hydrogel Matrix

The gel sheets were subsequently immersed in excess 0.1 dilution for 2 hours at room temperature, and DAB treat- M phosphate buffer solution for 24 h at 37°C to remove ment was carried out to develop brown color. Photomi- DMSO, and disc-shaped gels with 6 mm diameter were crographs of cross-sections were taken with a digital cut out from the gel sheets (1 mm thick) for in vivo microscope (BZ-9000, Keyence, Osaka, Japan) to ana- implantation. In the present study, we synthesized twenty lyze neovascular development in the implanted material. kinds of TSC-AlCol gels for evaluation (Table 1) and Since neovascularization in the material must progress abbreviated the TSC-AlCol gel with formulation X mM from the tissue surrounding the material, analysis of TSC and Y % AlCol to TSC[X]-AlCol[Y]. cross sections was restricted to the peripheral region of each material, which was within 200 μm from the over- Animal model and implantation of TSC-AlCol matrices all margin of the material profile, and the area of analy- Male Sprague-Dawley rats (3–4 months, Nisseizai, sis was first measured with Image J software (National Tokyo, Japan) fed a normal diet were used in all experi- Institutes of Health, USA). Another reason for setting the ments, and all protocols were conducted according to the analysis area within 200 μm from the margin was to Guide for Animal Experimentation of The University of eliminate the potential influence of a slight difference in Tokyo. Rats were anesthetized by intraperitoneal injec- implant shape and/or deformation. Then, using angiogen- tion of sodium pentobarbital (50 mg/kg body wt). The esis image analyzer software (KURABO, Kurabo, Osaka, rectus sheath was exposed through a middle incision of Japan), the digital data of photomicrographs were bina- the abdomen, a longitudinal fasciotomy of the rectus rized at a pre-set threshold, and the pixels that constituted sheath was carried out near the linea alba, and an appro- blood vessels were counted in each binarized image.17) priate space for implantation was prepared between the The images were furthermore converted into line images rectus sheath and the rectus abdominus muscle. After by a thinning process, and the length of vessels and the insertion of each TSC-AlCol gel disc (n = 6 for each gel number of vessel joints and branches were measured. disc) in the subfascial space, the rectus sheath was closed Parameters for neovascular development were calculated with polypropylene sutures. In another set of animals, a as follows. flat drop (30 μl, ca. 37 μg) of MatrigelTM (BD Biosci- Vessel density (pixels/μm2) = pixels of blood vessels / ences, CA, USA) was implanted in the same manner (n = analysis area 6). The volume of 30 μl MatrigelTM was almost the same Vessel length (μm/μm2) = length of vessels / analysis as that of the TSC-AlCol gel disc (6 mm diameter, 1 mm area thick), and the flat drop of MatrigelTM was arranged in a Vessel joints (/μm2) = number of vessel joints / analy- similar shape to that of the TSC-AlCol gel disc before sis area gelation. Vessel path (/μm2) = number of vessel branches / anal- At 7 days after implantation, these implants were ysis area excised with the surrounding rectus sheath and muscle Analysis of the images was repeated twice in a blinded tissue, fixed with 4% phosphate buffered paraformalde- manner by a student helper, and the mean of the three hyde (0.1 mol/l PO4 buffer, pH 7.3) overnight, and embed- values was used for statistical analysis. ded in paraffin. Combination of bFGF with TSC-Alcol gel Analysis of neovascular development in the TSC-Al- To evaluate what amount of bFGF potentially com- Col gel bines with the TSC-AlCol gel, the gels were incubated Each paraffin block was trimmed in order to expose with sufficient bFGF, and the amount of combined bFGF the maximal transverse profile of the implanted materi- was measured. An excess (1 μg) of human recombinant als, and 4-μm-thick cross-sections were cut. All sections bFGF (R&D Systems, MN, USA) was added to three were stained with hematoxylin & eosin. Then, other sets kinds of TSC-AlCol gel discs (6 mm diameter and 1 mm of cross-sections were stained for lectin to identify thick, n = 4 each) whose formulation was 3, 6 or 10mM endothelial cells lining blood vessels.16) The sections were TSC and 15% AlCol (TSC[3, 6, 10]-AlCol 15). After 3 first treated with 1% trypsin (Roche Applied Science) for hours of incubation in a humid chamber at room temper- 10 minutes, and then incubated with 3% hydrogen perox- ature, the gel discs were washed three times with PBS to ide for 10 minutes. Subsequently, biotinylated lectin remove uncombined bFGF. Then, the bFGF-treated gel (L5391, Sigma, St. Louis, MO, USA) was applied at a 1:50 disc was homogenized and lysed in 100 μl high-salt

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Nagayoshi M, et al. buffer (50mM Tris [pH7.6], 1.5M NaCl, 1 mM PMSF, 10 contrast, other TSC-AlCol gels and MatrigelTM main- μg/ml leupeptin), and the volume of the lysate was mea- tained their gel structure even at 7 days after implanta- sured (VLYSATE). The concentration of bFGF in the lysate tion. Regarding granulation growth into the residual

(CbFGF) was quantified by ELISA using a bFGF ELISA material, a certain amount of granulation tissue devel- kit (R&D Systems) according to the manufacturer’s oped with inflammatory infiltration in TSC [3]-AlCol [15, instructions, and the amount of bFGF bound to each gel 20, 30], TSC [6]-AlCol [7.5, 10, 15, 20, 30], TSC [10]-AlCol TM disc was calculated as VLYSATE × CbFGF. [7.5], and Matrigel , whereas little growth of granulation tissue was observed in TSC [10]-AlCol [10, 15, 20, Vascular development after implantation of bFGF- 30] (Fig. 2). treated TSC-AlCol gels Another series of TSC-AlCol gel discs (Fig. 1B) was Neovascular development after implantation of the incubated with 1 μg bFGF for 3 hours at room tempera- TSC-AlCol gel ture, and washed three times with PBS. The bFGF- Histological staining for lectin successfully demon- treated gel discs were implanted into the subfascial space strated the distribution of the vasculature in host tissue of the rat abdomen. At 7 days after implantation, the and also in the implanted materials. Vascular develop- implants were resected from the animals, and vascular ment into the implanted materials was detected in TSC development in the gels was evaluated in the same man- [3]-AlCol [15, 20, 30], TSC [6]-AlCol [7.5, 10, 15, 20, 30], ner. TSC [10]-AlCol [7.5], and MatrigelTM (Fig. 2). All parameters of neovascular development showed that values in Statistical analysis TSC [3]-AlCol [15], TSC [6]-AlCol [7.5, 10, 15] and TSC All data are presented as mean ± SE for each group. [10]-AlCol [7.5] were significantly higher than those in Student’s t-test was used for comparison of each group. P MatrigelTM (Fig. 3). values < 0.05 were considered statistically significant. Combination of bFGF with the TSC-AlCol gel Results Three kinds of TSC-AlCol gels (TSC [3, 6, 10]-AlCol [15]) were treated with a sufficient volume of bFGF, and Synthesis of TSC-AlCol gels ELISA analysis for bFGF showed that bFGF constantly The gels were successfully synthesized in the pre- bound to each TSC-AlCol gel. The amount of bFGF sented formulations (Table 1). Reaction solvent, DMSO, bound to TSC [3]-AlCol [15] was significantly greater was completely substituted by water. The color of the gels than that bound to TSC [6]-AlCol [15] and TSC [10]- was water-clear, and the shape and hardness of the gels AlCol [15], while there was no difference between the were stable in water for more than 1 month. As the con- values of TSC [6]-AlCol [15] and TSC [10]-AlCol [15] centration of TSC or AlCol decreased, the hardness of (Fig. 4). the synthesized gels became softer. In addition to the presented formulations, we tried to make TSC-AlCol Vascular development after implantation of bFGF- mixtures whose concentration of AlCol was 5% or less; treated TSC-AlCol gels however, these mixtures did not gelate, irrespective of The combination of bFGF significantly increased ves- AlCol concentration. sel density and length in TSC [3]-AlCol [15], TSC [6]- AlCol [7.5, 10] and TSC [10]-AlCol [7.5], as compared Histological findings of excised implants with those in untreated gels (Fig. 3A and 3B). Further, Histological analysis with hematoxylin & eosin stain- the vessel joints and path showed significantly higher ing showed the in vivo status of each material at 7 days values in bFGF-treated gels than in untreated plain gels after implantation, and also revealed how granulation (Fig. 3C and 3D). development and inflammatory reactions were induced in and around the material on the same day. After Discussion implantation of TSC [1]-AlCol [7.5, 10, 15, 20, 30] and TSC [3]-AlCol [7.5, 10] gels, the structure of the gels A scaffold structure is essential for induction of neo- completely disappeared, and no accumulation of inflam- vascular growth into regenerative tissues, since neovascu- matory cells was detected around the implanted site. In lature requires surrounding supportive tissues to maintain

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Neovascular Formation in Collagen Hydrogel Matrix

A B C D

E F G H Fig. 2 Photomicrographs of excised implants at 7 days after implantation into the rat subfascial space. Presented micrographs are from implants of Matrigel (A, E), TSC [10]-AlCol [10] (B, F), TSC [6]-AlCol [10] (C, G) and bFGF-combined TSC [6]-AlCol [10] (D, H). Sections were stained with hematoxylin & eosin (A, B, C, D), and also stained for lectin to detect vessels (E, F, G, H). There was no tissue growth into TSC [10]-AlCol [10] implants (B, F), whereas granulation and vessel growth (arrow) were noted in other implants. Bar: 50 μm

Fig. 3 Quantitative analysis of lectin staining. Vessel density, vessel length, vessel joints and vessel path in the part A B analyzed were measured. Values are shown as mean ± SD (n = 6). *P < 0.05 vs Matrigel, **P < 0.01 vs Matri- gel, #P < 0.05 non-treated gel vs bFGF-combined gel, ##P < 0.01 non-treated gel vs bFGF-combined gel. C D its luminal structure and biological performance.9) In the muscle. Although some prototypes induced no growth of present study, we developed a novel gel material that vasculature, gels with appropriate concentrations of TSC could function as a scaffold for neovascular growth. and AlCol markedly promoted neovascular formation in Many prototypes of hydrogel material were made by the gels at 7 days after implantation, which surpassed the incorporating several concentrations of TSC and AlCol, angiogenic ability of MatrigelTM. and implanted into the subfascial space of the rat rectus There are some possible reasons why TSC-AlCol gels

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newly formed vessels themselves occupy certain extents of the space. It is also important that the structure of the hydrogel contains cell adhesive sites to function as a scaffold for vascularized tissue. In vivo, ECM supports the vascular wall by anchoring vascular wall cells to matrix molecules. Attachment between vascular cells and surrounding ECM molecules is critical to establish the luminal structure of vessels and regulation of vessel growth.9) In the mechanism of cell-ECM attachment, integrins play an important role as cell surface receptors.18) Integrins are obligate heterodimers consisting of α and β subunits. In Fig. 4 Capacity of bFGF combination with TSC [10]-AlCol [15], mammals, 19 α and 8 β subunits have been character- TSC [6]-AlCol [15] and TSC [3]-AlCol [15] gel discs (6 ized, and more than 20 types of integrins with different mm diameter, 1 mm thick). An excess amount of bFGF combinations of α and β subunits have been identified.20) was added to each gel, and 3 hours later, combined Each integrin respectively binds with unique ECM com- bFGF was measured. Values are shown as mean ± SD (n = 4). *P < 0.05, NS: not significant. ponents. As a matter of course, endothelial and other vascular cells also express several kinds of integrins on their cell membrane,21) and some of these integrins can induced favorable growth of vascularized tissue into the directly bind with collagen.22) However, the predominant material. One crucial reason might be that the TSC- integrin-ligand in cell-ECM attachment is fibronectin, a AlCol gel provided a suitable matrix microstructure as a high-molecular-weight glycoprotein.23) Especially, platform for vessel formation. In vivo, vessels are natu- fibronection is known to be a potent ligand of αvβ3 and rally included in ECM, and ECM possesses the character αvβ5, which are critical integrins in neovascularization of a hydrogel. Thus, it is not surprising that artificial and granulation formation.21) Fibronection is abundantly hydrogel also reproduces a microstructure that mimics contained in blood plasma in its soluble form, and it is natural ECM.18) Indeed, Zisch et al. synthesized an artifi- also able to potently associate with collagen molecules.23) cial hydrogel material based on polyethylene glycol Therefore, in the in vivo condition, which is fibronectin- (PEG) and synthetic peptides, and showed that vascular- rich, integrins on the cell surface possibly bind with col- ized tissue grew into the hydrogel material after implan- lagen molecules through the intermediary of sufficient tation into the rat subcutis.19) To realize such ECM-mim- fibronectin. These properties of collagen molecules sug- icking properties, however, the polymer network of the gest that TSC-AlCol gels possess adequate cell adhesive artificial hydrogel must have an adequate density. If the sites in their structure to function as a scaffold. network of the polymer structure is too dense, vascular Another possible reason explaining the vasculariza- growth into the hydrogel could be physically blocked, tion ability of the TSC-AlCol gel might be that the and if the interstitial distance of the network is too large, appropriate part of the gel was degraded according to the microstructure of the hydrogel would not sustain the neovascular growth into its inside. In this regard, it is surrounding wall of the neovasculature as a scaffold. In critical that gel degradation should occur only in the the present study, no growth of vasculature was observed direction of neovascular growth, and the other part of in some prototypes, possibly indicating that their micro- the gel structure must be preserved as a scaffold for structure did not provide appropriate space for neovascu- newly formed vessels. For elongation and enlargement of lar formation. Further, the combination of bFGF signifi- vessels in vivo, vascular wall cells and infiltrating cells cantly promoted neovascular formation in some proto- in ECM generally release a variety of proteases, such as types, in which vascular development was seemingly matrix metalloproteinases (MMPs) and plasminogen observed more in the hydrogel containing less AlCol. It activators (PAs), and promote the degradation of a sur- possibly indicates that too dense TSC and AlCol would rounding ECM to ensure space for vessel growth.24, 25) interfere with vascularization especially in the bFGF Therefore, the scaffold material for ingrowth of vascu- treated hydrogels because once bFGF was impregnated larized tissue must contain protease-cleavable sites and larger quantity of vascular development occurred and concomitantly possess stability against variable local sit-

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Neovascular Formation in Collagen Hydrogel Matrix uations except angiogenic proteases. Collagen molecules process of arteriogenesis in addition to angiogenesis and/ contain abundant MMPs-cleavable sites,26) indicating that or vasculogenesis. Since bFGF is known to induce angio- collagen is a potential material for induction of neovas- genesis and arteriogenesis,6, 28) it is an ideal angiogenic cularization. Indeed, a previous in vitro study showed factor for increasing the effect of vascular development that tube formation of endothelial cells was accelerated in the TSC-AlCol gel. Further, it might be important that under culture in a gelated emulsion of collagen.27) This bFGF associated with the TSC-AlCol gel. When bFGF evidence demonstrated that gelated collagen was success- solution is added to the TSC-AlCol gel, bFGF molecules fully remodeled in vitro according to neovascularization, might permeate through the matrix structure of the TSC- and also functioned as a scaffold for newly formed ves- AlCol gel and then bind to the TSC part with ionic sels. However, since collagen molecules are merely bonds, because bFGF is positively charged and TSC is entangled in the emulsion form, a gelated emulsion of negatively charged.13) Therefore, there is a reserve of collagen might be dissolved by adding excessive fluid. If bFGF inside the TSC-AlCol gel, which stimulates the such a collagen material is implanted under physiological vessels that have grown into the gel, accelerating vascular conditions in vivo, exudates accumulated around the development inside the gel. The present study indeed implant consequentially dilute the collagen and then revealed that combination with bFGF significantly cause disintegration of its structure as a scaffold. There- increased vascular formation in the TSC-AlCol gel. Fur- fore, it is necessary to cross-link collagen molecules to ther, measurement of bFGF level also showed that the stabilize the gel structure. Since the TSC-AlCol gel is a concentration of combined bFGF in the TSC-AlCol gel chemical gel in which collagen molecules are cross- was reasonable for proliferation of ordinary cells, though linked by a citric acid derivative, TSC-AlCol gel enabled it might be necessary to clarify the relation between TSC maintenance of a scaffold structure for vascular tissue, concentration and combined bFGF level.29) If the TSC- while only the gel part in the direction of vessel growth AlCol gel is unable to bind with bFGF, bFGF in the gel was digested by released proteases. might spread to the outside after implantation, and neo- Further, the citric acid-based crosslinker, TSC also vascularization might be induced only around the gel. functions as a reservoir of bFGF. In general, angiogenic In summary, we created novel hydrogel materials by humoral factors play important roles in the induction of incorporating several concentrations of TSC and AlCol, neovascularization, and previous studies have demon- and an in vivo study using a rat model showed that some strated several growth factors and cytokines to be angio- of these materials had induced marked growth of vascu- genic factors.5) Each angiogenic factor possesses specific lature-rich tissue into the inside of the materials at 7 days functions that contribute to neovascularization, and after implantation. Further, the ability of vascular appropriate combinations of these functions promote ingrowth was significantly enhanced by combining appropriate vessel formation according to situational bFGF with the materials. These findings suggest that the demand. Indeed, there are three processes in the forma- hydrogel materials functioned as a favorable scaffold for tion of new vessels; vasculogenesis, angiogenesis and neovascular formation and also as a reservoir for con- arteriogenesis, and these processes are generally induced trolled delivery of bFGF. by distinctive combinations of angiogenic factors.28) Vas- culogenesis in the adult represents a process by which References endothelial progenitor cells differentiate and replicate to form a primitive network of vasculature, and angiogene- 1) MacNeil S. Progress and opportunities for tissue-engi- sis is the process of endothelial migration and replication neered skin. Nature 2007; 445: 874-80. resulting in the formation of a capillary network. In con- 2) Raghunath J, Rollo J, Sales KM, Butler PE, Seifalian AM. Biomaterials and scaffold design: key to tissue- trast, arteriogenesis refers to the maturation and enlarge- engineering cartilage. 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