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1.  Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor Mediated Augmentation of Angiogenesis and Bone Formation in Vascularized Bone Allotransplants 
Microsurgery  2014;34(4):301-307.
We previously demonstrated recipient-derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long-term immunosuppression. The effect of cytokine delivery to enhance this process is studied.
Vascularized femur transplantation was performed from DA to PVG rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer (N=11), basic fibroblast growth factor (FGF2) (N=10), vascular endothelial growth factor (VEGF) (N=11), or both (N=11) were inserted intramedullarly alongside a recipient-derived a/v bundle. FK-506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric and alkaline phosphatase measurements were performed.
Bone blood flow was greater in the combined group than control and VEGF groups (p=0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (p<0.05). Bone viability, growth and alkaline phosphatase activity did not vary significantly between groups.
Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants.
PMCID: PMC3976711  PMID: 24395434
2.  Cell lineage in vascularized bone transplantation 
Microsurgery  2013;34(1):37-43.
The biology behind vascularized bone allotransplantation remains largely unknown. We aim to study cell traffic between donor and recipient following bone auto,-and allografting.
Vascularized bone allografts and isografts were analyzed in a rat model at 4 and 18 weeks. Bone remodeling areas were laser capture microdissected. Analysis by RT-PCR measured the relative Expression Ratio (rER) of donor to recipient cells.
The rER was 0.456 (+/−0.266) at 4 weeks in allotransplants and 0.749 (+/−0.387) at 18 weeks, implying donor bone was gradually repopulated with recipient bone forming cells. In isotransplants, rER was 0.412 (+/−0.239) at 4 and 0.467 (+/−0.252) at 18 weeks. Cells in the inner and outer cortical bone remodeling areas were mainly donor derived (rER<0.5) at 18 weeks in isotransplants and mainly recipient derived in allotransplants (rER>0.5).
Applying novel methodology we describe detailed cell traffic in vascularized bone transplants, elaborating our comprehension on bone transplantation.
PMCID: PMC3972888  PMID: 24038399
3.  Effect of rhBMP-2 and VEGF in a Vascularized Bone Allotransplant Experimental Model Based on Surgical Neoangiogenesis 
We have demonstrated survival of living allogeneic bone without long-term immunosuppression using short-term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study bone morphogenic protein-2 (rhBMP-2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra-medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP-2 or rhBMP-2 + VEGF. FK-506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP-2 + VEGF) group than rhBMP-2 and control groups (p<0.05). Osteoblast counts were also higher in the rhBMP-2 + VEGF group (p<0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p=0.059). Local administration of VEGF and rhBMP-2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts.
PMCID: PMC3972920  PMID: 23192572
bone; allotransplantation; microspheres; BMP; VEGF
4.  Surgical angiogenesis with short-term immunosuppression maintains bone viability in rabbit allogenic knee joint transplantation 
Plastic and reconstructive surgery  2013;131(2):148e-157e.
Vascularized Composite Allotransplantation (VCA) has potential for reconstruction of joint defects, but requires life-long immunosuppression (IS), with substantial risks. This study evaluates an alternative, using surgical angiogenesis from implanted autogenous vessels to maintain viability without long-term immunotherapy.
Vascularized knee joints were transplanted from Dutch Belted donors to New Zealand White rabbit recipients. Once positioned and revascularized microsurgically, a recipient-derived superficial inferior epigastric fascial (SIEF) flap and a saphenous AV bundle were placed within the transplanted femur and tibia, respectively, to develop a neoangiogenic, autogenous circulation. Ten transplants comprised Group 1. Group 2 (n=9) were no-angiogenesis controls with ligated flaps and AV bundles. Group 3 rabbits (n=10) were autotransplants with patent implants. Tacrolimus was used for 3 weeks to maintain nutrient flow during angiogenesis. At 16 weeks, we assessed bone healing, joint function, bone and cartilage mechanical properties and histology.
Group 1 allotransplants had more robust angiogenesis, better healing, improved mechanical properties and better osteocyte viability than ligated controls (group 2). All 3 groups developed knee joint contractures and arthritic changes. Cartilage thickness and quality were poorer in allograft groups than autotransplant controls.
Surgical angiogenesis from implanted autogenous tissue improves bone viability, healing and material properties in rabbit allogenic knee transplants. However, joint contractures and degenerative changes occurred in all transplants, regardless of antigenicity or blood supply. Experimental studies in a larger animal model with improved methods to maintain joint mobility are needed before the merit of living joint allotransplantation can be judged.
PMCID: PMC3927985  PMID: 23358010
5.  Knee joint transplantation combined with surgical angiogenesis in rabbits – a new experimental model 
Microsurgery  2011;32(2):118-127.
We have previously described a means to maintain bone allotransplant viability, without long-term immune modulation, replacing allogenic bone vasculature with autogenous vessels. A rabbit model for whole knee joint transplantation was developed and tested using the same methodology, initially as an autotransplant.
Eight New Zealand White rabbit knee joints were elevated on a popliteal vessel pedicle to evaluate limb viability in a non-survival study. Ten additional joints were elevated and replaced orthotopically in a fashion identical to allotransplantation, obviating only microsurgical repairs and immunosuppression. A superficial inferior epigastric facial (SIEF) flap and a saphenous arteriovenous (AV) bundle were introduced into the femur and tibia respectively, generating a neoangiogenic bone circulation. In allogenic transplantation, this step maintains viability after cessation of immunosuppression. Sixteen weeks later, x-rays, microangiography, histology, histomorphometry and biomechanical analysis were performed.
Limb viability was preserved in the initial 8 animals. Both soft tissue and bone healing occurred in 10 orthotopic transplants. Surgical angiogenesis from the SIEF flap and AV bundle was always present. Bone and joint viability was maintained, with demonstrable new bone formation. Bone strength was less than the opposite side. Arthrosis and joint contractures were frequent.
We have developed a rabbit knee joint model and evaluation methods suitable for subsequent studies of whole joint allotransplantation.
PMCID: PMC3321575  PMID: 22113889
Microsurgery  2010;30(7):557-564.
Previous papers have shown surgical neoangiogenesis to allow long-term bone allotransplant survival without immunosuppression. Whole joint composite tissue allotransplants (CTA) might be treated similarly. A novel rat knee CTA model is described for further study of the roles of neoangiogensis in joint allotransplant survival and adjustment of immunosuppression.
Microvascular knee CTA was performed in 9 rats across a major histocompatibility barrier with both pedicle repair and implantation of host-derived arteriovenous (‘a/v’) bundles. In the control group (N=3), the pedicle was ligated. Immunosuppression was given daily. Joint mobility, weight-bearing, pedicle patency, bone blood flow and sprouting from a/v bundles were assessed at three weeks.
All but the non-revascularized control knees had full passive motion and full weight bearing. One nutrient pedicle thrombosed prematurely. Blood flow was measurable in transplants with patent nutrient pedicles. Implanted a/v bundles produced new vascular networks on angiography.
This new rat microsurgical model permits further study of joint allotransplantation. Patency of both pedicles and implanted a/v bundles was maintained, laying a foundation for future studies.
PMCID: PMC2956790  PMID: 20842706
7.  Augmentation of Surgical Angiogenesis in Vascularized Bone Allotransplants with Host-Derived AV Bundle Implantation, Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor Administration 
We have previously shown experimental transplantation of living allogeneic bone to be feasible without long-term immunosuppression by development of a recipient-derived neoangiogenic circulation within bone. In this study we study the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from DA to PVG rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both were inserted intramedullarly along with a recipient-derived a/v bundle. FK-506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic and histomorphometric analysis were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p<0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p<0.005). Bone formation rates were greater with VEGF (p<0.01) and FGF+VEGF (p<0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression.
PMCID: PMC2892011  PMID: 20162714
bone; allotransplantation; microspheres; FGF; VEGF
8.  Repopulation of vascularized bone allotransplants with recipient-derived cells: Detection by laser capture microdissection and real-time PCR 
Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation.
Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. 4 groups differed in use of immunosuppression (+/- 2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene.
Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.
PMCID: PMC2872153  PMID: 19437510
microdissection; bone; allotransplant; real-time PCR; chimerism

Results 1-8 (8)