Indian Journal of Plastic Surgery
An open access publication of Association of Plastic Surgeons of India
Users Online: 605  
Home | Subscribe | Feedback | Login 
  Navigate here 
  Search
 
  
 Resource links
 »   Similar in PUBMED
 »  Search Pubmed for
 »  Search in Google Scholar for
 »Related articles
 »   Article in PDF (3,389 KB)
 »   Citation Manager
 »   Access Statistics
 »   Reader Comments
 »   Email Alert *
 »   Add to My List *
* Registration required (free)  
  In this article
 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 »  References
 »  Article Figures
 »  Article Tables

 Article Access Statistics
    Viewed2518    
    Printed61    
    Emailed0    
    PDF Downloaded54    
    Comments [Add]    
    Cited by others 2    

Recommend this journal

 


 
 Table of Contents    
ORIGINAL ARTICLE
Year : 2012  |  Volume : 45  |  Issue : 3  |  Page : 444-452
 

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


1 Department of Plastic and Reconstructive Surgery and Burn Unit, Gülhane Military Medical Academy and Medical Faculty, Haydarpasa Training Hospital, Istanbul, Turkey
2 Department of Genetics and Bioengineering, Yeditepe University, Faculty of Engineering and Architecture, Istanbul, Turkey
3 Department of Nuclear Medicine, Gülhane Military Medical Academy and Medical Faculty, Haydarpasa Training Hospital, Istanbul, Turkey
4 Department of Pathology, Gülhane Military Medical Academy and Medical Faculty, Haydarpasa Training Hospital, Istanbul, Turkey

Date of Web Publication12-Jan-2013

Correspondence Address:
Celalettin Sever
Department of Plastic and Reconstructive Surgery and Burn Unit, Gulhane Military Medical Academy, Haydarpasa Training Hospital, Selimiye Mahallesi Tibbiye Caddesi 34668, Uskudar, Istanbul
Turkey
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-0358.105939

Rights and Permissions

 » 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.


Keywords: Bone prefabrication; interconnected porous calcium hydroxyapatite ceramic; rat bone marrow mesenchymal stem cells; vascular endothelial growth factor


How to cite this article:
Sever C, Uygur F, Kose GT, Urhan M, Haholu A, Kulahci Y, Sinan O, Cihan S, Omer O. 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. Indian J Plast Surg 2012;45:444-52

How to cite this URL:
Sever C, Uygur F, Kose GT, Urhan M, Haholu A, Kulahci Y, Sinan O, Cihan S, Omer O. 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. Indian J Plast Surg [serial online] 2012 [cited 2019 Apr 18];45:444-52. Available from: http://www.ijps.org/text.asp?2012/45/3/444/105939



 » Introduction Top


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. [1],[2]

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. [3],[4] 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. [5],[6]

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],[8],[9],[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],[12],[13],[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. [7],[8],[11],[12],[13],[14],[15],[16],[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 Top


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 1]a. 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 1]b. Biocompatible PCHC blocks were shaped to form cylindrical shapes [Figure 1]c. 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 1]d.
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

Click here to view


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 × 10 6 cells/ml. [1],[15] Subsequently, mesenchymal stem cells (5 × 10 6 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

Click here to view


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

Click here to view


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

Click here to view
Figure 4: The superficial epigastric artery and vein in the tunnel of the PCHC block

Click here to view
Figure 5: The PCHC block without vascular bundle implantation

Click here to view


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

Click here to view


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 45 th day. Each PCHC was crushed, homogenized in 0.2% Nonidet P-40/50 mM Tris-HCl buffer containing 1 mM MgCl 2 , 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 Top


Microangiography

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

Click here to view


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

Click here to view
Figure 9: Uptake of radioactivity

Click here to view


Biochemical analysis

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

Click here to view


Histologic evaluation

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

Click here to view



 » Discussion Top


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. [1],[5] 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. [5],[16]

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. [1],[17],[18] The most popular three-dimensional scaffold is PCHC. [1],[5],[7],[10] 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. [1],[18] 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. [1],[21],[22],[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],[29],[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. [7],[8] 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.

 
 » References Top

1.Sever C, Uygur F, Külahci Y, Torun Köse G, Urhan M, Kuçukodaci Z, et al. Effect of hyperbaric oxygen therapy on bone prefabrication in rats. Acta Orthop Traumatol Turc 2010;44:403-9.  Back to cited text no. 1
    
2.Horch RE, Kopp J, Kneser U, Beier J, Bach AD, Tissue engineering of cultured skin substitutes. J Cell Mol Med 2005;9:592-608.  Back to cited text no. 2
    
3.Weiland AJ, Moore JR, Daniel RK. Vascularized bone autografts. Experience with 41 cases. Clin Orthop Relat Res 1983;174:87-95.  Back to cited text no. 3
[PUBMED]    
4.More JB, Mazur JM, Zehr D, Davis PK, Zook EG. A biomechanical comparison of vascularized and conventional autogenous bone grafts. Plast Reconstr Surg 1984;73:382-6.  Back to cited text no. 4
    
5.Di Bella C, Lucarelli E, Donati D. Historical review of bone prefabrication. Chir Organi Mov 2008;92:73-8.  Back to cited text no. 5
[PUBMED]    
6.Khouri RK, Upton J, Shaw WW. Principles of flap prefabrication. Clin Plast Surg 1992;19:763-71.  Back to cited text no. 6
[PUBMED]    
7.Yang P, Wang C, Shi Z, Huang X, Dang X, Li X, et al. Rh VEGF 165 delivered in a porous beta-tricalcium phosphate scaffold accelerates bridging of critical-sized defects in rabbit radii. J Biomed Mater Res A 2010;92:626-40.  Back to cited text no. 7
[PUBMED]    
8.Yang P, Wang C, Shi Z, Huang X, Dang X, Xu S, et al. Prefabrication of vascularized porous three-dimensional scaffold induced from rh VEGF (165): A preliminary study in rats. Cells Tissues Organs 2009;189:327-37.  Back to cited text no. 8
[PUBMED]    
9.Kaigler D, Wang Z, Horger K, Mooney DJ, Krebsbach PH. VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects. J Bone Miner Res 2006;21:735-44.  Back to cited text no. 9
[PUBMED]    
10.Tamai N, Myoui A, Tomita T, Nakase T, Tanaka J, Ochi T, et al. Novel hydroxyapatite ceramics with an interconnective porous structure exhibit superior osteoconduction in vivo. J Biomed Mater Res 2002;59:110-7.  Back to cited text no. 10
[PUBMED]    
11.Dai J, Rabie AB. VEGF: An essential mediator of both angiogenesis and endochondral ossification. J Dent Res 2007;86:937-50.  Back to cited text no. 11
[PUBMED]    
12.Bates DO, Jones RO. The role of vascular endothelial growth factor in wound healing. Int J Low Extrem Wounds 2003;2:107-20.  Back to cited text no. 12
[PUBMED]    
13.Peng HR, Wright V, Usas A, Gearhart B, Shen HC, Cummins J, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest 2002;110:751-9.  Back to cited text no. 13
    
14.Leach JK, Kaigler D, Wang Z, Krebsbach PH, Mooney DJ. Coating of VEGF releasing scaffolds with bioactive glass for angiogenesis and bone regeneration. Biomaterials 2006;27:3249-55.  Back to cited text no. 14
[PUBMED]    
15.Zhao Z, Yang D, Ma X, Zhao H, Nie C, Si Z. Successful repair of a critical-sized bone defect in the rat femur with a newly developed external fixator. Tohoku J Exp Med 2009;219:115-20.  Back to cited text no. 15
[PUBMED]    
16.Gill DR, Ireland DC, Hurley JV, Morrison WA. The prefabrication of a bone graft in a rat model. J Hand Surg Am 1998;23:312-21.  Back to cited text no. 16
[PUBMED]    
17.Fujibayashi S, Kim HM, Neo M, Uchida M, Kokubo T, Nakamura T. Repair of segmental long bone defect in rabbit femur using bioactive titanium cylindrical mesh cage. Biomaterials 2003;24:3445-51.  Back to cited text no. 17
[PUBMED]    
18.Yoshikawa H, Tamai N, Murase T, Myoui A. Interconnected porous hydroxyapatite ceramics for bone tissue engineering. J R Soc Interface 2009;6:S341-8.  Back to cited text no. 18
[PUBMED]    
19.Khaled EG, Saleh M, Hindocha S, Griffin M, Khan WS. Tissue engineering for bone production-stem cells, gene therapy and scaffolds. Open Orthop J 2011;5(Suppl 2):289-95.  Back to cited text no. 19
[PUBMED]    
20.Javerzat S, Auguste P, Bikfalvi A. The role of fibroblast growth factors in vascular development. Trends Mol Med 2002;8:483-9.  Back to cited text no. 20
[PUBMED]    
21.Sata M, Tanaka K, Nagai R. Circulating osteoblast-lineage cells. N Engl J Med 2005;353:737-8.  Back to cited text no. 21
[PUBMED]    
22.Roufosse CA, Direkze NC, Otto WR, Wright NA. Circulating mesenchymal stem cells. Int J Biochem Cell Biol 2004;36:585-97.  Back to cited text no. 22
[PUBMED]    
23.Otsuru S, Tamai K, Yamazaki T, Yoshikawa H, Kaneda Y. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice. Biochem Biophys Res Commun 2007;354:453-8.  Back to cited text no. 23
[PUBMED]    
24.Mizumoto S, Inada Y, Weiland AJ. Fabrication of vascularized bone grafts using ceramic chambers. J Reconstr Microsurg 1993;9:441-9.  Back to cited text no. 24
[PUBMED]    
25.Nettelblad H, Randolph MA, Ostrup LT, Weiland AJ. Molded vascularized osteoneogenesis: A preliminary study in rabbits. Plast Reconstr Surg 1985;76:851-8.  Back to cited text no. 25
[PUBMED]    
26.Akita S, Tamai N, Myoui A, Nishikawa M, Kaito T, Takaoka K, et al. Capillary vessel network integration by inserting a vascular pedicle enhances bone formation in tissue-engineered bone using interconnected porous hydroxyapatite ceramics. Tissue Eng 2004;10:789-95.  Back to cited text no. 26
[PUBMED]    
27.Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: The potential for engineering bone. Eur Cell Mater 2008;15:100-14.  Back to cited text no. 27
[PUBMED]    
28.Geiger F, Bertram H, Berger I, Lorenz H, Wall O, Eckhardt C, et al. Vascular endothelial growth factor gene-activated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects. J Bone Miner Res 2005;20:2028-35.  Back to cited text no. 28
[PUBMED]    
29.Street J, Winter D, Wang JH, Wakai A, McGuinness A, Redmond HP. Is human fracture hematoma inherently angiogenic? Clin Orthop Relat Res 2000;378:224-37.  Back to cited text no. 29
[PUBMED]    
30.Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 1999;5:623-8.  Back to cited text no. 30
[PUBMED]    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2]


This article has been cited by
1 Vascularization in Bone Tissue Engineering Constructs
Ángel E. Mercado-Pagán,Alexander M. Stahl,Yaser Shanjani,Yunzhi Yang
Annals of Biomedical Engineering. 2015;
[Pubmed] | [DOI]
2 prefabrication of vascularized grafts based on pre-differentiated adipose derived stem cells, fibrin sealant and porous calcium phosphate cement scaffold
yang, p. and huang, x. and wang, c.-s. and wang, k.-z.
chinese journal of tissue engineering research. 2013; 17(51): 8801-8808
[Pubmed]



 

Top
Print this article  Email this article
 

    

Site Map  |  Home  |  Contact Us  |  Feedback  |  Copyright and Disclaimer
Online since 11th March '04
Published by Wolters Kluwer - Medknow