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author:("Larsen, mirko")
1.  Cell lineage in vascularized bone transplantation 
Microsurgery  2013;34(1):37-43.
Background
The biology behind vascularized bone allotransplantation remains largely unknown. We aim to study cell traffic between donor and recipient following bone auto,-and allografting.
Methods
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.
Results
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).
Conclusion
Applying novel methodology we describe detailed cell traffic in vascularized bone transplants, elaborating our comprehension on bone transplantation.
doi:10.1002/micr.22147
PMCID: PMC3972888  PMID: 24038399
2.  A MODIFIED VASCULARIZED WHOLE KNEE JOINT ALLOTRANSPLANTATION MODEL IN THE RAT 
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.
doi:10.1002/micr.20800
PMCID: PMC2956790  PMID: 20842706
3.  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.
doi:10.1002/jor.21098
PMCID: PMC2892011  PMID: 20162714
bone; allotransplantation; microspheres; FGF; VEGF
4.  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.
doi:10.1002/jor.20915
PMCID: PMC2872153  PMID: 19437510
microdissection; bone; allotransplant; real-time PCR; chimerism

Results 1-4 (4)