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.

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.

INTRODUCTION

Bone defects caused by trauma, infection, resection of tumors represent a major problem in plastic surgery and traumatology. Vascularized bone flaps and autogenous bone grafts are being used to reconstruct bone defects presently, because of their obvious advantages in osteogenic potential, mechanical properties and the lack of adverse immunological response.[12]

Autogenous bone grafting has been the gold standard for reconstruction of bone defects. However, autogenous bone grafting has some limitations, such as donor site morbidity, and inadequate availability of grafts in the requisite size and shape. Otherwise, allografts carry the risk of infectious diseases; the sterilization procedures applied to prevent infectious diseases adversely affect the biological properties of allografts.[34] Therefore, demineralized bone matrices such as bioactive glass and ceramic biomaterials are being produced as alternatives to autografts and allografts.[1] Using these materials, it is possible to reconstruct tissues, such as bone, cartilage, muscle, or skin in shapes and sizes that can replace nearly every defect, while ensuring minimum morbidity in the donor site and improving the reconstruction efficacy markedly.[56]

In order to establish a three dimensional bone tissue structure, there has to be a skeleton system, i.e., a cell carrier for the osteoblasts to hold on to and proliferate. The most popular for bone prefabrication among these systems is PCHC. It is essential that PCHC is bio-compatible, so that it enables root cells to stick to each other and proliferate. PCHC should also include a porous structure that facilitates vascular structures to proceed inward. There are more than 90% pores in calcium hydroxyapatite ceramic blocks and since these pores are in touch with each other, PCHC is the most preferred bio-material for bone prefabrication.[7-10]

VEGF is critical in angiogenesis and it is responsible for endothelial cell proliferation and migration. Controlled delivery of both angiogenic and osteogenic growth factors may mimic natural bone healing to promote the regeneration of critical size of bone defects.[11-14] In literature, there are many experimental and clinical studies related to bone prefabrication and prelamination. In these studies, both bone growth factors and cytokines have been used for bone prefabrication.[7811-17] The present study demonstrated that vascularization of interconnected PCHC by vascular bundle insertion, along with a combination of VEGF microspheres and mesenchymal stem cells increases new bone formation as well as capillary vessel formation with up-regulated expression of VEGF. However, further studies using clinically relevant animal models are needed to assess the potential role of mesenchymal stem cells and VEGF. The purpose of this experimental study is to investigate the impact of mesenchymal stem cells and VEGF on bone prefabrication.

MATERIALS AND METHODS

This study was reviewed and approved by the Ethics Committee for Experimental Animals of Haydarpasa Numune Education and Research Hospital. In this study, sixty male Sprague-Dawley rats were used. Experimental animals were divided into six groups, each of which comprised 10 rats.

All the PCHC blocks (Pro Osteon® 500R Porous Bone Graft Substitute, Interpore Cross International, Irvine, CA, USA) were sterilized in the autoclave before use. They were made up of spherical pores of uniform size and almost all of them were connected through interconnected holes. They had the following characteristics: Porosity of 90% and an average pore size of 180 μm. The majority of the interpore connections ranged from 10-80 μm in diameter, which would theoretically allow cell migration or tissue invasion from pore to pore [Figure 1a]. In the first phase of this study, the structural view of PCHC blocks was evaluated by scanning electron microscopy (Carl Zeiss SMT Inc. Thornwood, NY) [Figure 1b]. Biocompatible PCHC blocks were shaped to form cylindrical shapes [Figure 1c]. A total of 60 cylinder shaped PCHC blocks 0.8 cm in length and 0.6 cm in width were obtained. A tunnel of 0.2 cm diameter was formed inside these blocks, using a drilling machine with a hollow drill [Figure 1d].

Figure 1

(a) The interconnected porous calcium hydroxyapatite ceramic 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 hydroxyapatite ceramic block

In the second phase of this study, mesenchymal stem cells were aspirated from the femurs of rats. These stem cells were cultured in media, which included 100 μg/ml penicillin and 25 μg/ml gentamycin at 37°C. After 14 days, the cultured mesenchymal stem cells were separated with trypsinogen and resuspended to 3 × 106 cells/ml.[115] Subsequently, mesenchymal stem cells (5 × 106 cell/ml) were implanted into each one of the cylinder shaped PCHC blocks in groups III, IV, V and VI. A solution containing dexamethasone (0.1 microns), sodium beta-glycerophosphate (10mM), vitamin C phosphate (80 mg/ml) was prepared to encourage osteogenic differentiation of mesenchymal stem cells.[1] PCHC blocks were kept in this solution for two days [Figure 2].

Figure 2

PCHC blocks in media for osteogenic differentiation

In the third phase of the study, recombinant human VEGF165 (rhVEGF165) was obtained from PeproTech EC (London, U.K.). One microgram of VEGF165 was added to 500 μl of an organic solution of 500 mg PLGA. Microspheres of PLGA incorporating VEGF 165 (VEGF microspheres) were thus prepared. The VEGF microspheres were applied to the PCHC in groups V and VI before the surgical procedure. The details of the six experimental goups are given in Table 1.

Table 1

The details of the six experimental groups

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 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 the distal end to its origin. The superficial inferior epigastric artery and vein were passed through the hole of the PCHC blocks in groups II, IV and VI [Figure 4]. The PCHC blocks were transplanted in the medial thigh region in groups I, III, and V without vascular bundle implantation [Figure 5].

Figure 3

The superficial epigastric artery and vein

Figure 4

The superficial epigastric artery and vein in the tunnel of the PCHC block

Figure 5

The PCHC block without vascular bundle implantation

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].

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

The presence and density of any new bone formation and neovascularization from the vascular bundle was evaluated by microangiography, scintigraphy, biochemical analysis and histomorphometry.

Evaluation methods

Scintigraphy

Bone scintigraphy was used for the evaluation of neovascularization and osteoblastic activity in the PCHC vascularized blocks on day 30. Technetium (Tc), marked with 99m methylene diphosphonate (Tc-99 mMDP) was used as the radiopharmaceutical agent. Two microcurie of Tc-99 mMDP was infused to the left jugular vein of rat on day 30 in all groups and the spot images were obtained after 2 hours (static images were taken with a 256 × 256 matrix zoom for 5 minutes). Blastic activity of the PCHC block was measured and compared with the measured blastic activity of symmetrical right thigh.[1]

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]

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 Biotech AB, Uppsala, Sweden) and 10% formic acid. 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).

Statistical evaluation

The Mann-Whitney U test was used for the evaluation and comparison of all test groups. All values were obtained using SPSS 10.0 software (SPSS Inc, Chicago, USA). The mean standard deviation and skewness values in each group were all compared due to the limited number of rats in test groups. The reliability of the Mann-Whitney U test was checked in this manner.

RESULTS

The degree of neovascularization in vascularized blocks in groups II, IV and VI was assessed by microangiography. There was no neovascularization in group I. In group II, neovascularization was seen only in the center of PCHC ceramic blocks. However, neovascularization starting from the center of PCHC ceramics and extending to the periphery was observed in group VI. Therefore, maximum neovascularization was clearly detected in group VI [Figure 7].

Figure 7

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

Scintigraphy

Radioactivity was detected only in groups II, IV and VI. There was no radioactivity in the other groups. However, the ratio of radioactivity in group VI was higher than groups II and IV [Figure 8]. For quantitative evaluation of bone scintigraphy, the uptake of radioactivity of PCHC blocks in each group was compared with the symmetric soft tissue of the PCHC blocks and the average values of these three groups were taken. According to the Mann-Whitney U test, the radioactivity uptake in group VI was higher than that in groups II and VI and this difference was statistically significant (P < 0.05) [Figure 9].

Figure 8

The radioactivity uptake rate of the PCHC blocks

Figure 9

Uptake of radioactivity

The levels of osteocalcin and alkaline phosphatase in PCHC blocks were measured. The average level of osteocalcin and alkaline phosphatase in group VI was higher than other groups [Table 2]. This difference was statistically significant (P < 0.05).

Table 2

The mean level of osteocalcin and alkaline phosphatase in PCHC blocks in all groups

Histologic evaluation

Maximum bone formation and neovascularization was observed in group VI during histological evaluation too [Figure 10].

Figure 10

Bone formation and neovascularization

DISCUSSION

Tissue engineering is a new field of biotechnology that focuses on the development of biological equivalents in order to repair or replace damaged tissue. It is one of the most interesting areas of plastic and reconstructive surgery because it represents a bridge between conventional reconstructive surgery and tissue engineering.[15] The studies in tissue engineering so far promise great hope for the future.

Reconstructing bone defects with autogenous bone grafting or free vascularized bone flap are useful techniques. However, these techniques are limited by the number of available donor sites, donor site morbidity, and the difficulty of microsurgical techniques. Therefore, bone prefabrication appears to be one of the most interesting and useful areas of reconstructive surgery. The term ‘prefabricated’ indicates a process of neovascularization of the tissue by implanting a vascular pedicle inside the tissue itself. After a period, this tissue has its own vascularization and it may be reimplanted either at a short distance through the pedicle itself, or as a free graft by microvascular anastomosis.[516]

Bone prefabrication requires three elements: A three-dimensional scaffold, blood supply, and finally a stimulus, through growth factors or stromal mesenchymal stem cells, which are specific for the tissue. An important point of discussion, presently, concerns the most convenient type of scaffold to be used.[5] Therefore, many kinds of biomaterials have been developed as bone substitutes, such as hydroxyapatite, alumina, polymers, metal, bioglasses, and organic or inorganic bone substitutes.[11718] The most popular three-dimensional scaffold is PCHC.[15710] There are more than 90% pores in calcium hydroxyapatite ceramic blocks. Scanning electron microscopy analysis revealed that most of the PCHC pores were spherical, similar in size, approximately 100-300 μm in diameter, and showed uniform connections with one another. The interconnected porous structure of PCHC facilitates bone prefabrication by allowing the introduction of mesenchymal cells, osteotropic agents or vasculature into the pores.[118] In addition, PCHC may be shaped according to the defect size and is inexpensive and readily available. All materials have the advantage 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. Although, the main source of mesenchymal stem cells is the bone marrow, they may also be isolated from various other tissues such as muscle, bone, cartilage, fat, liver, cord blood, peripheral blood and fetal bone. Stem cells regenerate themselves by dividing, can form tissues that serve specialized purposes and have differentiation abilities. The physiological function of adult stem cells is to facilitate tissue homeostasis and tissue regeneration after injury. In in vitro conditions, stem cells may transform to different cell types such as adipogenic, myogenic and osteogenic cells. However, the specifics of the differentiation mechanism of stem cells and progenitor cells are still unknown.[121-23]

Experimental and clinical studies indicate that vascularized biomaterials improved osteocyte survival and enhanced bone incorporation. Mizumoto et al. reported that vascular bundle implantation increased early bone formation, with neovascularization, in a hydroxyapatite scaffold with bone marrow cells.[24] Nettelblad et al. reported molded vascularized osteogenesis, using titanium chambers with autogenous corticocancellous bone chips and vascular bundle implantation.[25] Sever et al. reported that vascular bundle implantation increased early bone formation with neovascularization in a hydroxyapatite scaffold with mesenchymal stem cells under hyperbaric oxygen therapy.[1] Akita et al. demonstrated bone formation by inserting a vascular bundle into PCHC loaded with recombinant bone morphogenetic protein-2.[26]

For a scaffold of customized vascularized bone graft, PCHC is considered to be one of the best materials in our study. The cultured mesenchymal stem cells were implanted into the PCHC blocks because PCHC blocks are biocompatible and osteoconductive. The implantation of a vascular bundle (superficial inferior epigastric artery and vein) and cultured mesenchymal stem cells with VEGF administration into PCHC caused good penetration of newly formed vessels and osteoid formation. Alkaline phosphatase activity and osteocalcin levels are the two most important biochemical components for the assessment of bone prefabrication. Alkaline phosphatase is located in the cell membranes of osteoblasts and therefore, it has a direct correlation with osteoblast activity. Osteocalcin is synthesized by osteoblasts and is the key factor pointing to the presence of bone tissue.[1] In our study, the highest levels of alkaline phosphatase and osteocalcin have been observed in group VI. The vascularization of PCHC blocks is necessary for the stem cells to survive and proliferate. VEGF administration enhanced the number and length of newly formed vessels at 4 weeks after surgery, and osteoid deposition was observed at 6 weeks.

VEGF is a potent angiogenic factor, essential for both intramembranous and endochondral bone formation. Intramembraneous ossification is characterized by invasion of capillaries into the mesenchymal zone, and differentiation of mesenchymal cells to osteoblasts. This type of ossification occurs during embryonic development. It is involved in the development of flat bones in the cranium and parts of the mandible. Endochondral ossification utilizes the functional properties of cartilage and bone to provide a mechanism for this formation.[27] VEGF is essential in coordinating metaphyseal and epiphyseal vascularization, cartilage formation, and ossification. VEGF stimulates migration and proliferation of vascular cells for vascular remodeling.[28-30] There is a cross-talk between endothelial cells and osteoblasts in which VEGF plays a key role: Osteoblast-like cells produce VEGF while VEGF enhances osteoblast differentiation.[28] VEGF induces differentiation of osteoblasts around the newly formed vessels, facilitated by the migration and proliferation of mesenchymal stem cells. Therefore, it is reasonable to suppose that the combination of VEGF, mesenchymal stem cells and vascular bundle implantation could enhance angiogenesis and bone formation in PCHC.

This study demonstrated that vascularization of PCHC by vascular bundle insertion along with a combination of VEGF microspheres and mesenchymal stem cells could increase new bone formation and capillary vessel formation with up-regulated expression of VEGF. Although the message and conclusion of the experimental studies by Yang et al.[78] are similar to our study, there are specific differences between the two. Firstly, Yang et al. administered only VEGF into the scaffolds encapsulated with muscle flap and saphenous vessels. In the present study, both mesenchymal stem cells and VEGF were administered into the scaffolds. The insertion of the vascular bundle in the PCHC block in our experiment is another point of difference. Furthermore, we show that application of VEGF along with the mesenchymal stem cells is more efficient in bone prefabrication.

Bone formation and osseointegration of biomaterials are dependent on angiogenesis and vascularization. The mesenchymal stem cells and VEGF microspheres act as positive stimulating factors and present a fast and effective way to promote osteogenesis and angiogenesis in bone prefabrication. Enhancing our understanding of the interaction of bone vascular biology and osteogenesis with further investigations will ultimately increase our knowledge and help in the design of novel and rational strategies for bone prefabrication.