Original Article

Prefabrication of vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic in presence of vascular endothelial growth factor and bone marrow mesenchymal stem cells: Experimental study in rats

Celalettin Sever, Fatih Uygur, Gamze Torun Kose1, Muammer Urhan2, Abdullah Haholu3, Yalcin Kulahci, Oksuz Sinan, Sahin Cihan, Ozcan Omer2 Department of Plastic and Reconstructive Surgery and Burn Unit, Gülhane Military Medical Academy and Medical Faculty, Haydarpasa Training Hospital, 1Genetics and Bioengineering, Yeditepe University, Faculty of Engineering and Architecture, 2Nuclear Medicine, 3Pathology, Gülhane Military Medical Academy and Medical Faculty, Haydarpasa Training Hospital, Istanbul, Turkey

Address for correspondence: Dr. Celalettin Sever, Department of Plastic and Reconstructive Surgery and Burn Unit, Gulhane Military Medical Academy, Haydarpasa Training Hospital, Selimiye Mahallesi Tıbbiye Caddesi 34668, Uskudar, Istanbul, Turkey. E‑mail: [email protected]

ABSTRACT

Objectives: The purpose of this experimental pilot study was to create a prefabricated vascularized bone graft using interconnected porous calcium hydroxyapatite ceramic (PCHC) block by combining vascular bundle implantation, rat bone marrow mesenchymal stem cells and administration of vascular endothelial growth factor (VEGF) in a rat model. Materials and Methods: Sixty male Sprague‑Dawley rats were used. Experimental animals were divided into six groups, each of which comprised 10 rats. The PCHC blocks were implanted in the medial thigh region in groups I, III, and V without vascular bundle implantation. The PCHC blocks were vascularized by the superficial inferior epigastric artery and vein in groups II, IV and VI. These vessels were passed through the hole of the PCHC blocks. Mesenchymal stem cells were administered into the PCHC in groups III, IV, V and VI. In addition, both mesenchymal stem cells and VEGF were administered in group V and VI. The presence and density of any new bone formation and neovascularization from the vascular bundle was evaluated by X‑ray, microangiography, scintigraphy, biochemical analysis and histomorphometry. Results: The newly formed vessels and bone formations were significantly greater in group VI, in which both mesenchymal stem cells and VEGF were applied. Conclusion: This preliminary study suggests that: Both mesenchymal stem cells and VEGF provide vascularized bone prefabrication by enhancing neovascularization and osteogenesis in a shorter time compared to only VEGF application.

Access this article online Quick Response Code: Website: KEY WORDS www.ijps.org Bone prefabrication; interconnected DOI: porous calcium hydroxyapatite ceramic; 10.4103/0970-0358.105939 rat bone marrow mesenchymal stem cells; vascular endothelial growth factor

Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 444 Sever, et al.: Prefabrication of vascularized bone graft

INTRODUCTION demonstrated that vascularization of interconnected PCHC by vascular bundle insertion, along with a combination of one defects caused by trauma, infection, resection VEGF microspheres and mesenchymal stem cells increases of tumors represent a major problem in plastic new bone formation as well as capillary vessel formation Bsurgery and traumatology. Vascularized bone with up‑regulated expression of VEGF. However, further flaps and autogenous bone grafts are being used to studies using clinically relevant animal models are needed reconstruct bone defects presently, because of their to assess the potential role of mesenchymal stem cells obvious advantages in osteogenic potential, mechanical and VEGF. The purpose of this experimental study is to properties and the lack of adverse immunological investigate the impact of mesenchymal stem cells and response.[1,2] VEGF on bone prefabrication.

Autogenous bone grafting has been the gold standard MATERIALS AND METHODS for reconstruction of bone defects. However, autogenous bone grafting has some limitations, such as donor site This study was reviewed and approved by the morbidity, and inadequate availability of grafts in the Ethics Committee for Experimental Animals of requisite size and shape. Otherwise, allografts carry the Haydarpasa Numune Education and Research Hospital. risk of infectious diseases; the sterilization procedures In this study, sixty male Sprague‑Dawley rats were used. applied to prevent infectious diseases adversely affect Experimental animals were divided into six groups, each the biological properties of allografts.[3,4] Therefore, of which comprised 10 rats. demineralized bone matrices such as bioactive glass and ceramic biomaterials are being produced as alternatives All the PCHC blocks (Pro Osteon® 500R Porous Bone to autografts and allografts.[1] Using these materials, it is Graft Substitute, Interpore Cross International, Irvine, possible to reconstruct tissues, such as bone, cartilage, CA, USA) were sterilized in the autoclave before muscle, or skin in shapes and sizes that can replace nearly use. They were made up of spherical pores of every defect, while ensuring minimum morbidity in the uniform size and almost all of them were connected donor site and improving the reconstruction efficacy through interconnected holes. They had the markedly.[5,6] following characteristics: Porosity of 90% and an average pore size of 180 µm. The majority of the interpore In order to establish a three dimensional bone tissue connections ranged from 10‑80 µm in diameter, which structure, there has to be a skeleton system, i.e., a cell would theoretically allow cell migration or tissue carrier for the osteoblasts to hold on to and proliferate. invasion from pore to pore [Figure 1a]. In the first The most popular for bone prefabrication among phase of this study, the structural view of PCHC blocks these systems is PCHC. It is essential that PCHC is was evaluated by scanning electron microscopy (Carl bio‑compatible, so that it enables root cells to stick to Zeiss SMT Inc. Thornwood, NY) [Figure 1b]. each other and proliferate. PCHC should also include a Biocompatible PCHC blocks were shaped to form porous structure that facilitates vascular structures cylindrical shapes [Figure 1c]. A total of 60 cylinder to proceed inward. There are more than 90% pores in shaped PCHC blocks 0.8 cm in length and 0.6 cm in calcium hydroxyapatite ceramic blocks and since these width were obtained. A tunnel of 0.2 cm diameter was pores are in touch with each other, PCHC is the most formed inside these blocks, using a drilling machine preferred bio‑material for bone prefabrication.[7‑10] with a hollow drill [Figure 1d].

VEGF is critical in angiogenesis and it is responsible for In the second phase of this study, mesenchymal stem endothelial cell proliferation and migration. Controlled cells were aspirated from the femurs of rats. These stem delivery of both angiogenic and osteogenic growth cells were cultured in media, which included 100 µg/ml factors may mimic natural bone healing to promote penicillin and 25 µg/ml gentamycin at 37°C. After 14 days, the regeneration of critical size of bone defects.[11‑14] In the cultured mesenchymal stem cells were separated literature, there are many experimental and clinical studies with trypsinogen and resuspended to 3 × 106 cells/ related to bone prefabrication and prelamination. In these ml.[1,15] Subsequently, mesenchymal stem cells (5 × 106 studies, both bone growth factors and cytokines have been cell/ml) were implanted into each one of the cylinder used for bone prefabrication.[7,8,11‑17] The present study shaped PCHC blocks in groups III, IV, V and VI. A solution

445 Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 Sever, et al.: Prefabrication of vascularized bone graft containing dexamethasone (0.1 microns), sodium Evaluation methods beta‑glycerophosphate (10mM), vitamin C phosphate Scintigraphy (80 mg/ml) was prepared to encourage osteogenic Bone scintigraphy was used for the evaluation differentiation of mesenchymal stem cells.[1] PCHC blocks of neovascularization and osteoblastic activity in the PCHC were kept in this solution for two days [Figure 2]. vascularized blocks on day 30. Technetium (Tc), marked with 99m methylene diphosphonate (Tc‑99 mMDP) was In the third phase of the study, recombinant human used as the radiopharmaceutical agent. Two microcurie VEGF165 (rhVEGF165) was obtained from PeproTech EC (London, U.K.). One microgram of VEGF165 was Table 1: The details of the six experimental groups added to 500 µl of an organic solution of 500 mg Groups Mesenchymal stem cells Vascular bundle VEGF PLGA. Microspheres of PLGA incorporating VEGF I − − − 165 (VEGF microspheres) were thus prepared. The VEGF II − + − III + − − microspheres were applied to the PCHC in groups V and IV + + − VI before the surgical procedure. The details of the six V + − + experimental goups are given in Table 1. VI + + +

Surgical procedure In the surgical phase of this study, anesthesia was induced by intramuscular injection of ketamine, at a dose of 40 mg/kg body weight, and sodium pentobarbital injection, at a dose of 10 mg/kg body weight. Inguinal areas were washed with 10% povidone‑iodine solution. The hair on the rat’s lower extremity was removed with a razor. Under sterile conditions, a transverse incision was made over a b the anterior side of left thigh. Subsequently, the superficial inferior epigastric artery and vein were reached by blunt and sharp dissections using a surgical microscope [Figure 3]. When the vascular bundle had been completely freed from the surrounding tissue, the distal end of this bundle was ligated and elevated from c d the distal end to its origin. The superficial inferior Figure 1: (a) The interconnected porous calcium hydroxyapatite ceramic epigastric artery and vein were passed through the hole block (b) PCHC image under scanning electron microscopy (c) PCHC blocks, 0.8 cm long and 0.6 cm wide (d) A tunnel with a 0.2 cm diameter inside the of the PCHC blocks in groups II, IV and VI [Figure 4]. hydroxyapatite ceramic block The PCHC blocks were transplanted in the medial thigh region in groups I, III, and V without vascular bundle implantation [Figure 5].

The second phase of the surgical procedure was performed two weeks later in all groups. PCHC blocks were reached by reopening the incision line. PCHC blocks in all groups were covered with silicone to prevent vascular invasion from the surrounding tissue [Figure 6].

The presence and density of any new bone formation and neovascularization from the vascular bundle was evaluated by microangiography, scintigraphy, biochemical analysis and histomorphometry. Figure 2: PCHC blocks in media for osteogenic differentiation

Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 446 Sever, et al.: Prefabrication of vascularized bone graft of Tc‑99 mMDP was infused to the left jugular vein of rat Statistical evaluation on day 30 in all groups and the spot images were obtained The Mann‑Whitney U test was used for the evaluation and after 2 hours (static images were taken with a 256 × 256 comparison of all test groups. All values were obtained matrix zoom for 5 minutes). Blastic activity of the PCHC using SPSS 10.0 software (SPSS Inc, Chicago, USA). The block was measured and compared with the measured mean standard deviation and skewness values in each [1] blastic activity of symmetrical right thigh. group were all compared due to the limited number of

Microangiography Microangiography was applied to five rats in vascularized blocks in groups II, IV and VI on day 35. The left carotid arteries of rats were exposed by a left oblique cervical incision. The proximal segment of the carotid artery was cannulated with a 20G epidural catheter after connecting the distal segment of carotid artery. One ml (5000 IU) heparin (Nevparin 5000 IU/ml) was injected. Fifteen minutes later, a solution prepared with lactated Ringer (75 ml), barium sulfate (20 ml) and bovine gelatin (5%) was heated up to 36°C and the catheter was infused with this solution using a low‑pressure technique. After the infusion was complete, the rats were kept in the refrigerator at 4°C. The radiographs were taken using mammography (at 23 KV, 12 mAs dose) after 12 hours.[1] Figure 3: The superficial epigastric artery and vein

Biochemical analysis Assays of alkaline phosphatase activity and osteocalcin content were carried out according to our previous report.[1] The osteocalcin and alkaline phosphatase levels of PCHC blocks in all groups were measured utilizing biochemical methods on the 45th day. Each PCHC was crushed, homogenized in 0.2% Nonidet

P‑40/50 mM Tris‑HCl buffer containing 1 mM MgCl2, and centrifuged at 13,000g for 15 minutes at 4°C. The osteocalcin activity was measured using a Sephadex

G‑25 column (NAP‑25 column, Amersham Pharmacia Figure 4: The superficial epigastric artery and vein in the tunnel of the PCHC Biotech AB, Uppsala, Sweden) and 10% formic acid. block The alkaline phosphatase activity was measured using ‘p‑nitrophenyl phosphate.[1]

Histological examination After the tissue samples were taken on day 45, PCHC was fixed in 10% formalin at 4°C for 24 hours. Subsequently, tissue samples were washed with distilled water and decalcified by leaving them in nitric acid for 72 hours. Five micrometer pathological sections were obtained from paraffin embedded blocks after the decalcification. These samples were stained with hematoxylin eosin (HE).[1] On each slide, six standardized regions of interest (ROIs) were photographed at a × 10 magnification with the Axioplan 2 (Carl Zeiss, Oberkochen, Germany). Figure 5: The PCHC block without vascular bundle implantation

447 Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 Sever, et al.: Prefabrication of vascularized bone graft rats in test groups. The reliability of the Mann‑Whitney U the ratio of radioactivity in group VI was higher than test was checked in this manner. groups II and IV [Figure 8]. For quantitative evaluation of bone scintigraphy, the uptake of radioactivity of PCHC RESULTS blocks in each group was compared with the symmetric soft tissue of the PCHC blocks and the average values Microangiography of these three groups were taken. According to the The degree of neovascularization in vascularized blocks Mann‑Whitney U test, the radioactivity uptake in group VI in groups II, IV and VI was assessed by microangiography. was higher than that in groups II and VI and this difference There was no neovascularization in group I. In group II, was statistically significant (P < 0.05) [Figure 9]. neovascularization was seen only in the center of PCHC ceramic blocks. However, neovascularization starting Biochemical analysis from the center of PCHC ceramics and extending to The levels of osteocalcin and alkaline phosphatase the periphery was observed in group VI. Therefore, in PCHC blocks were measured. The average level of maximum neovascularization was clearly detected in osteocalcin and alkaline phosphatase in group VI was group VI [Figure 7]. higher than other groups [Table 2]. This difference was statistically significant (P < 0.05). Scintigraphy Radioactivity was detected only in groups II, IV and VI. Histologic evaluation There was no radioactivity in the other groups. However, Maximum bone formation and neovascularization was

a b c Figure 6: PCHC blocks were reached by reopening the incision line (a) Pedicled PCHC block after two weeks (b) Nonpedicled PCHC block after two weeks (c) PCHC blocks in all groups were covered with silicone to prevent vascular invasion from the surrounding tissue

Figure 7: The microangiographic views of PCHC in vascularized groups II, IV and VI

Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 448 Sever, et al.: Prefabrication of vascularized bone graft observed in group VI during histological evaluation either at a short distance through the pedicle itself, or as too [Figure 10]. a free graft by microvascular anastomosis.[5,16]

DISCUSSION Bone prefabrication requires three elements: A three‑dimensional scaffold, blood supply, and Tissue engineering is a new field of biotechnology that finally a stimulus, through growth factors or stromal focuses on the development of biological equivalents in mesenchymal stem cells, which are specific for the tissue. order to repair or replace damaged tissue. It is one of the An important point of discussion, presently, concerns the most interesting areas of plastic and reconstructive surgery most convenient type of scaffold to be used.[5] Therefore, because it represents a bridge between conventional many kinds of biomaterials have been developed as bone reconstructive surgery and tissue engineering.[1,5] The substitutes, such as hydroxyapatite, alumina, polymers, studies in tissue engineering so far promise great hope metal, bioglasses, and organic or inorganic bone for the future. substitutes.[1,17,18] The most popular three‑dimensional scaffold is PCHC.[1,5,7,10] There are more than 90% pores Reconstructing bone defects with autogenous bone in calcium hydroxyapatite ceramic blocks. Scanning grafting or free vascularized bone flap are useful electron microscopy analysis revealed that most of the techniques. However, these techniques are limited PCHC pores were spherical, similar in size, approximately by the number of available donor sites, donor site 100‑300 µm in diameter, and showed uniform connections morbidity, and the difficulty of microsurgical techniques. with one another. The interconnected porous structure Therefore, bone prefabrication appears to be one of of PCHC facilitates bone prefabrication by allowing the the most interesting and useful areas of reconstructive introduction of mesenchymal cells, osteotropic agents or surgery. The term ‘prefabricated’ indicates a process of vasculature into the pores.[1,18] In addition, PCHC may be neovascularization of the tissue by implanting a vascular shaped according to the defect size and is inexpensive pedicle inside the tissue itself. After a period, this tissue and readily available. All materials have the advantage has its own vascularization and it may be reimplanted of unlimited availability and good osteoconductive properties. But, they are not osteoinductive, thus limiting their application in the repair of large bone defects.[19] For this reason, PCHC has also been used as a composite with stem cells or cytokines.[20]

The basic structure of tissue engineering is the stem cell. Bone marrow includes hemopoetic stem cells that develop into all blood cells as well as mesenchymal stem cells which are capable of forming connective tissue.

Figure 8: The radioactivity uptake rate of the PCHC blocks Figure 9: Uptake of radioactivity

Table 2: The mean level of osteocalcin and alkaline phosphatase in PCHC blocks in all groups Group I Group II Group III Group IV Group V Group VI Alkaline phosphatase (U/l) 0 0 30 200 140 440 Osteocalcin (ng/ml) 0 0 10 50 30 80

449 Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 Sever, et al.: Prefabrication of vascularized bone graft

Figure 10: Bone formation and neovascularization

Although, the main source of mesenchymal stem cells with mesenchymal stem cells under hyperbaric oxygen is the bone marrow, they may also be isolated from therapy.[1] Akita et al. demonstrated bone formation various other tissues such as muscle, bone, cartilage, by inserting a vascular bundle into PCHC loaded with fat, liver, cord blood, peripheral blood and fetal bone. recombinant bone morphogenetic protein‑2.[26] Stem cells regenerate themselves by dividing, can form tissues that serve specialized purposes and have For a scaffold of customized vascularized bone graft, differentiation abilities. The physiological function of PCHC is considered to be one of the best materials in adult stem cells is to facilitate tissue homeostasis and our study. The cultured mesenchymal stem cells were tissue regeneration after injury. In in vitro conditions, implanted into the PCHC blocks because PCHC blocks are stem cells may transform to different cell types such as biocompatible and osteoconductive. The implantation adipogenic, myogenic and osteogenic cells. However, the of a vascular bundle (superficial inferior epigastric artery specifics of the differentiation mechanism of stem cells and vein) and cultured mesenchymal stem cells with and progenitor cells are still unknown.[1,21‑23] VEGF administration into PCHC caused good penetration of newly formed vessels and osteoid formation. Alkaline Experimental and clinical studies indicate that vascularized phosphatase activity and osteocalcin levels are the two most biomaterials improved osteocyte survival and enhanced important biochemical components for the assessment of bone incorporation. Mizumoto et al. reported that vascular bone prefabrication. Alkaline phosphatase is located in bundle implantation increased early bone formation, the cell membranes of osteoblasts and therefore, it has a with neovascularization, in a hydroxyapatite scaffold with direct correlation with osteoblast activity. Osteocalcin is bone marrow cells.[24] Nettelblad et al. reported molded synthesized by osteoblasts and is the key factor pointing vascularized osteogenesis, using titanium chambers with to the presence of bone tissue.[1] In our study, the highest autogenous corticocancellous bone chips and vascular levels of alkaline phosphatase and osteocalcin have been bundle implantation.[25] Sever et al. reported that vascular observed in group VI. The vascularization of PCHC blocks bundle implantation increased early bone formation is necessary for the stem cells to survive and proliferate. with neovascularization in a hydroxyapatite scaffold VEGF administration enhanced the number and length

Indian Journal of Plastic Surgery September-December 2012 Vol 45 Issue 3 450 Sever, et al.: Prefabrication of vascularized bone graft of newly formed vessels at 4 weeks after surgery, and understanding of the interaction of bone vascular osteoid deposition was observed at 6 weeks. biology and osteogenesis with further investigations will ultimately increase our knowledge and help in VEGF is a potent angiogenic factor, essential for both the design of novel and rational strategies for bone intramembranous and endochondral bone formation. prefabrication. Intramembraneous ossification is characterized by invasion of capillaries into the mesenchymal zone, and REFERENCES differentiation of mesenchymal cells to osteoblasts. This type of ossification occurs during embryonic development. 1. Sever C, Uygur F, Külahci Y, Torun Köse G, Urhan M, It is involved in the development of flat bones in the cranium Kuçukodaci Z, et al. Effect of hyperbaric oxygen therapy on bone prefabrication in rats. 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