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1.  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
2.  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
3.  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
4.  Surgical Revascularization Induces Angiogenesis in Orthotopic Bone Allograft 
Remodeling of structural bone allografts relies on adequate revascularization, which can theoretically be induced by surgical revascularization. We developed a new orthotopic animal model to determine the technical feasibility of axial arteriovenous bundle implantation and resultant angiogenesis.
We asked whether arteriovenous bundles implanted in segmental allografts would increase cortical blood flow and angiogenesis compared to nonrevascularized frozen bone allografts and contralateral femoral controls.
We performed segmental femoral allotransplantation orthotopically from 10 Brown Norway rats to 20 Lewis rats. Ten rats each received either bone allograft reconstruction alone (Group I) or allograft combined with an intramedullary saphenous arteriovenous flap (Group II). At 16 weeks, we measured cortical blood flow with the hydrogen washout method. We then quantified angiogenesis using capillary density and micro-CT vessel volume measurements.
All arteriovenous bundles were patent. Group II had higher mean blood flow (0.12 mL/minute/100 g versus 0.05 mL/minute/100 g), mean capillary density (23.6% versus 2.8%), and micro-CT vessel volume (0.37 mm3 versus 0.07 mm3) than Group I. Revascularized allografts had higher capillary density than untreated contralateral femora, while vessel volume did not differ and blood flow was lower.
Axial surgical revascularization in orthotopic allotransplants can achieve strong angiogenesis and increases cortical bone blood flow.
Clinical Relevance
Poor allograft revascularization results in frequent complications of nonunion, infection, and late stress fracture. The presented technique of surgical revascularization could therefore offer a beneficial adjunct to clinical segmental bone allografting.
PMCID: PMC3830091  PMID: 22723247
5.  Partial Tibial Nerve Transfer to the Tibialis Anterior Motor Branch to Treat Peroneal Nerve Injury After Knee Trauma 
Injuries to the deep peroneal nerve result in tibialis anterior muscle paralysis and associated loss of ankle dorsiflexion. Nerve grafting of peroneal nerve injuries has led to poor function; therefore, tendon transfers and ankle-foot orthotics have been the standard treatment for foot drop.
We (1) describe an alternative surgical technique to obtain ankle dorsiflexion by partial tibial nerve transfer to the motor branch of the tibialis anterior muscle; (2) evaluate ankle dorsiflexion strength using British Medical Research Council grading after nerve transfer; and (3) qualitatively determine factors that influence functional success of surgery.
We retrospectively reviewed 11 patients treated with partial tibial nerve transfers after peroneal nerve injury. Pre- and postoperative motor strength was measured. Patients completed questionnaires regarding pre- and postoperative gait and disability.
One patient regained Grade 4 ankle dorsiflexion, three patients regained Grade 3, one patient regained Grade 2, and two patients regained Grade 1 ankle dorsiflexion. Four patients did not regain any muscle activity. Clinically apparent motor recovery occurred an average 7.6 months postoperatively. A majority of patients (nine) could walk and participate in activities. Seven patients did not wear ankle-foot orthotics and four patients did not limp. The donor deficits included weak toe flexion (two patients) and reduced calf circumference (seven patients).
Our observations suggest nerve transfers to the deep peroneal nerve provide inconsistent ankle dorsiflexion strength, possibly related to the mechanism of peroneal nerve injury or delays in surgery. Despite variable strength, four patients achieved M3 or greater motor recovery, which enabled them to walk without assistive devices.
Level of Evidence
Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC3270157  PMID: 21626085
6.  New workhorse flaps in hand reconstruction 
Hand (New York, N.Y.)  2012;7(1):45-54.
With the passage of time, certain hand surgery procedures are anecdotally dubbed “workhorse” techniques. These are procedures that are extremely reliable and have repeatedly demonstrated good results. However, with time, paradigms undergo shifts, and this is as true for hand surgery as any other field. In this article, we will describe the use of three new “workhorse” flaps that we have found to have reliable results in complex hand reconstruction: the pedicled radial forearm fascia flap for dorsal hand reconstruction, the free anterolateral thigh flap for mangled hand reconstruction, and the medial femoral condyle vascularized bone graft for scaphoid fracture nonunion reconstruction.
PMCID: PMC3280375  PMID: 23449685
Flap; Radial forearm; Anterolateral thigh; Femoral condyle
7.  A New Approach to Assess the Gastrocnemius Muscle Volume in Rodents Using Ultrasound; Comparison with the Gastrocnemius Muscle Index 
PLoS ONE  2013;8(1):e54041.
The purpose of this study was to determine the reliability and validity of a new non-invasive ultrasound technique to measure gastrocnemius muscle atrophy after nerve denervation in an animal model.
In sixteen rodents an eight mm sciatic nerve gap was created. In the following 8 weeks, each week, two rodents were euthanized and the gastrocnemius muscle was examined using two different ultrasound systems and two investigators. The standardized ultrasound measurement protocol consisted of identifying pre-defined anatomical landmarks: 1) the fibula, 2) the fibular nerve, and 3) the junction between the most distal point of the semitendinosus muscle and gastrocnemius muscle. Consequently, we measured the muscle thickness as the length of the line between the fibula and the junction between the two muscles, perpendicular to the fibular nerve. After the ultrasound recording, the muscle mass was determined.
A steep decline of muscle weight of 24% was observed after one week. In the following weeks, the weight further decreased and then remained stable from 6 weeks onwards, resulting in a maximal muscle weight decrease of 82%. The correlation coefficient was >0.96 between muscle diameter and weight using both ultrasound systems. The inter-rater reliability was excellent for both devices on the operated side (ICC of 0.99 for both ultrasound systems) and good for the non-operated site (ICC’s: 0.84 & 0.89). The difference between the muscle mass ratio and the muscle thickness ratio was not more than 5% with two outliers of approximately 13%.
We have developed an innovative, highly reliable technique for quantifying muscle atrophy after nerve injury. This technique allows serial measurements in the same animal over time. This is a significant advantage compared to the conventional technique for quantifying muscle atrophy, which requires sacrificing the animal.
PMCID: PMC3542319  PMID: 23326570
8.  Vascularized Bone Grafting in a Canine Carpal Avascular Necrosis Model 
Limited experimental research has been performed on the treatment of avascular necrosis (AVN) by vascularized bone grafting.
A new model simulating carpal AVN was created to investigate surgical revascularization of necrotic bone.
In seven mongrel dogs, AVN was induced by removal of the radial carpal bones bilaterally, deep-freezing, coating in cyanoacrylate, and reimplantation. A reverse-flow vascularized bone graft from the distal radius was implanted in the avascular radial carpal bone. The contralateral side served as an untreated ischemic control. Bone blood flow, bone volume, radiography, histomorphometry, histology, and MRI were analyzed at 4 weeks.
Blood flow was substantially higher in grafted bones when compared with controls (14.68 ± 15.43 versus 0.27 ± 0.28 mL/minute/100 g). Blood flow correlated with increased osteoid formation and higher levels of bone turnover. T1 and T2 signals on MRI did not correlate with quantitative bone blood flow measurements. Necrotic bones with no blood flow had normal T1 and T2 signals, whereas revascularized bones had signal changes when compared with adjacent carpal bones. No major collapse occurred in any radiocarpal bone.
In a canine experimental model, investigation of carpal AVN shows the ability of vascularized bone grafting to revascularize and remodel avascular bone.
Clinical Relevance
Surgical revascularization of necrotic bone induced by vascularized bone grafting results in increased bone perfusion and bone remodeling as compared with untreated necrotic bone. MRI T1 and T2 signals can be normal in necrotic avascular bone.
PMCID: PMC3171535  PMID: 21533527
9.  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
11.  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
12.  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
13.  Reverse Radial Forearm Fascial Flap With Radial Artery Preservation 
Hand (New York, N.Y.)  2007;2(3):159-163.
The reverse radial forearm fascial (RRFF) flap is widely used in soft-tissue reconstruction of the hand. The traditional RRFF flap incorporates the radial artery from the forearm and is perfused by retrograde flow through the palmar arch. In patients with an abnormal Allen test because of an incomplete palmar arch, the traditional RRFF flap is contraindicated unless a vein graft is used to reconstruct the radial artery. A simpler alternative approach for hand reconstruction in such patients is a distally based RRFF flap based on radial artery perforators, which preserves the radial artery. We used RRFF flaps based on radial artery perforators in five patients who had palmar or dorsal soft-tissue loss. All five recovered full hand function, and only one had any complications (full-thickness skin graft loss at recipient site). The RRFF flap based on distal radial artery perforators is suitable for thin coverage of soft-tissue defects in hands with either a complete or an incomplete palmar arch.
PMCID: PMC2527146  PMID: 18780079
Artery; Radial; Flap; Forearm fascial; Reconstruction; Soft-tissue

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