Quantifying Massive Allograft Healing of the Canine Femur In Vivo and Ex Vivo: A Pilot Study

Background

Allograft integration in segmental osseous defects is unpredictable. Imaging techniques have not been applied to investigate angiogenesis and bone formation during allograft healing in a large-animal model.

Questions/purposes

We used dynamic contrast-enhanced (DCE)-MRI and cone beam (CB)-CT to quantify vascularity and bone volume in a canine femoral allograft model and determined their relationship with biomechanical testing and histomorphometry.

Methods

Femoral ostectomy was performed in three dogs and reconstructed with a 5-cm allograft and compression plate. At 0.5, 3, and 6 months, we performed DCE-MRI to quantify vascular permeability (Ktrans) and perfused fraction and CB-CT to quantify bone volume. We also performed posteuthanasia torsional testing and dynamic histomorphometry of the grafted and nonoperated femurs.

Results

DCE-MRI confirmed the avascular nature of allograft healing (perfused fraction, 2.08%–3.25%). CB-CT demonstrated new bone formation at 3 months (26.2, 3.7, and 2.2 cm³) at the graft-host junctions, which remodeled down at 6 months (14.0, 2.2, and 2.0 cm³). The increased bone volume in one subject was confirmed with elevated Ktrans (0.22) at 3 months. CB-CT-identified remodeled bone at 6 months was corroborated by histomorphometry. Allografted femurs recovered only 40% of their strength at 6 months.

Conclusions

CB-CT and DCE-MRI can discriminate differences in angiogenesis and bone formation in the canine allograft model, which has potential to detect a small (32%) drug or device effect on biomechanical healing with only five animals per group.

Clinical Relevance

These radiographic tools may have the potential to assess adjuvant effects on vascular invasion and new bone formation after segmental allograft transplantation.

     

Wound Healing Modulators in a Tracheoplasty Canine Model

Postsurgical tracheal stenosis results from fibrosis formation due to ischemia. There are healing modulators, hyaluronic acid (HA) and collagen polyvinylpyrrolidone (CPVP), which reduce collagen fibers formation. Thus we can hypothesize that the topical application of one of these modulators can diminish postsurgical tracheal scarring and stenosis. The aim of this work was to evaluate the macroscopic, microscopic, and biochemical changes of tracheal healing after the application of HA or CPVP in a canine tracheoplasty model. The study design was prospective experimental investigation in a canine model. Eighteen mongrel dogs underwent three cervical tracheal rings resection and end-to-end anastomosis. They were randomized into three groups according to treatment: group I (control group) (n = 6), topical application of saline solution on tracheal anastomosis; group II (n = 6), topical application of 15 μ g HA on tracheal anastomosis; and group III (n = 6), topical application of 2.5 mg CPVP on tracheal anastomosis. They were evaluated clinical, radiological and tracheoscopically during 4 weeks. They were euthanized at the end of the study time. Macroscopic, microscopic, and biochemical changes of tracheal anastomosis healing were analyzed. Collagen formation was quantified by the Woessner method. All the animals survived the surgical procedure and study period. Macroscopic, radiologic, and endoscopic studies showed that animals in group I developed tracheal stenosis, inflammation, and firm fibrous tissue formation, and histological studies also showed severe inflammatory reaction and fibrosis formation. Groups II (HA) and III (CPVP) showed well-organized thin collagen fibers with minimal inflammatory response. Biochemical evaluation revealed a higher collagen concentration in group I animals (analysis of variance [ANOVA] p <. 05 and Tukey p <. 01). Thus, hyaluronic acid or collagen polyvinylpyrrolidone administered after tracheal anastomosis diminished the degree of stenosis and inflammatory reaction. Both modulators improved tracheal healing.     

A New Experimental Model for Simultaneous Evaluation of Aortic and Pulmonary Allograft Performance in a Composite Graft

A model was developed in pigs for simultaneous evaluation of aortic and pulmonary allograft performance in a composite graft. The composite graft consisted of vascular prosthesis and aortic and pulmonary allografts. Following antibiotic preservation, it was anastomosed to the recipient's thoracic descending aorta by an extrapleural approach without using cardiopulmonary bypass. Aortic blood flow was completely diverted into the composite graft. All 12 recipient pigs recovered well, 4 of which were assigned for the initial study to design the suitable experimental schedule. Calcification readily occurred in the aortic allografts and aneurysmal dilatation without calcvication developed in the pulmonary allografts. These morphological findings were consistent with those of previous reports. This model has several benefits. First, aortic and pulmonary allograft conduits can be implanted and evaluated simultaneously under the same conditions by making a composite graft. Second, the magnitude of the operation is minimum, and postoperative circulatory and respiratory management is uncomplicated. Third, wound infection rarely occurs, because the skin incision is made on the back. These preliminary studies suggest that this model will allow future study concerning aortic and pulmonary allograft conduits under different conditions     

Accelerated Endothelialization Model for the Study of Dacron Graft Healing

p < 0.05). The neointima, which contained smooth muscle cells, was formed as early as 2 weeks in the vein-wrapped grafts. There were no differences in the speed of healing or in healing patterns according to whether the intimal or the adventitial side of the inferior vena cava was placed against the graft. Histologic findings did not support the hypothesis that accelerated flow surface endothelialization results in direct migration of endothelial cells from the intima of the vein wrap, and there was no clear correlation between the surface endothelial-like cell coverage and microostia. To gain further insight into why accelerated healing occurs in this model, earlier observations accompanied by molecular biology analysis are needed, and vein wrap studies provide a method of comparison for this work.     

Effects of Caffeine Consumption on Autologous Full-Thickness Skin Graft Healing in an Animal Model

Background There exists contradictory evidence that states both the beneficial and deleterious effects of caffeine on wound healing. The general population might unknowingly consume caffeine that negatively affects wound healing. The main objective of this study is to investigate the effect of daily caffeine consumption on wound healing, specifically full-thickness skin graft (FTSG). Methods Forty Sprague–Dawley rats were randomized into four groups of equal size: control-dose (CD), low-dose (LD), medium-dose (MD), and high-dose (HD) caffeine groups. After autologous FTSG, all subjects in the intervention group were given daily pure caffeine gavage. The FTSG was explanted 7 days posttransplant. The graft viability, secondary contraction, and adherence were evaluated macroscopically, while fibroblast and collagen deposition was analyzed microscopically with hematoxylin eosin stain. Results The least graft viability (72.8 ± 20.7%, clinical wound assessment scale [CWAS] 2.4), highest secondary contraction (11.4 ± 10.5%), and fibroblast count (331.8 ± 88.6 cells/5 high power fields) were observed in the MD group. More collagen synthesis was observed in subjects who consumed caffeine. The level of secondary contraction, fibroblast count as well as graft viability and collagen synthesis were positively correlated. Conclusions Daily consumption of caffeine impairs graft viability when given in medium dose and increases collagen synthesis, irrespective of dosage. This study was in experimental rats; the results are not directly translatable to humans.     

Allograft Conduit Wall Calcification in a Model of Chronic Arterial Graft Rejection

A bstract Early allograft vascular wall degeneration has emerged as a major important complication in young patients. To explain this mechanism, we reviewed studies on explants of allograft valved conduits implanted heterotopically into the infrarenal aorta in inbred rats (LEW;RT1, and CAP-RT1°C). The following strain combinations (isografts and allografts) were used: syngeneic, LEW-> LEW, strongly allogeneic, and CAP > LEW (RT1- and non-RT1-incompatible). Second-set skin grafting was performed 3 weeks after the heterotrophic implant to test for immunogenicity and presensitization. The animals (LEW) were sacrificed serially on days 20, 30, 50, and 100 for immunofluorescence and SEM studies. Endothelial disruption was observed on day 30, while valve leaflets appeared normal. Humoral allograft rejection was demonstrated and associated with production of antibodies (IgG) against the endothelial cells and around the smooth muscle cells, and in areas of smooth cell necrosis, through 100 days. Neointimal repopulation by host cells and migrated smooth muscle cells was also observed in both viable and allovital grafts. Allovital grafts demonstrated more disorganized collagen and elastic fibers, as well as calcific degeneration in the media and neointima on day 50; the viable conduits showed such structural changes on day 100. In conclusion, vascular walls of allovital conduits calcified earlier than the viable conduits without discernible calcification of the valves. There is therefore evidence to prove causative relationships between cellular viability, immune response, and fibroproliferative calcific degeneration in allograft vascular conduits.     

异体DBM复合rhBMP-2修复兔桡骨缺损的实验研究

目的探讨异体脱钙骨基质（demineralized bonematrix，DBM）复合重组人骨形态发生蛋白2（reconstruction humn bonemorphology protein-2，rhBMP-2）修复节段性骨缺损的能力。方法48只新西兰大白兔采用桡骨15mm节段性骨缺损模型，随机分为3组，A组植入异体DBM与rhBMP-2复合材料，B组植入异体DBM，C组为空白对照组。术后4周、8周、12周、16周．进行放射学和组织学检查。结果A组：术后4周宿主结缔组织长入植骨材料内的骨小梁间，并有岛状新生软骨、骨组织形成；术后8周，新生软骨及骨形成并融合成片；术后12周，新骨改建成熟，但仍能见到植骨材料；术后16周，管状骨结构形成，髓腔再通。B组：术后4周，植骨材料周围有软骨形成；术后8周，大量软骨形成；术后12周，大片状骨形成；术后16周，有髓腔形成。C组：各时问点仅见有纤维结缔组织，只在两端有新骨形成。X片示A组成骨量大，新骨改建、成熟迅速，术后16周全部达骨性愈合。B组成骨量少，仅2例达骨性愈合。C组未见骨性愈合。结论异体DBM复合rhBMP-2材料通过骨诱导和骨传导两种方式修复骨缺损，是一种较理想、具有高效成骨活性的植骨材料。     

Healing of the Canine Aorta After Endarterectomy: A Scanning Electron Microscopy Study

Endarterectomy sections of canine aorta were examined in various stages of healing by scanning electron microscopy. In all specimens the endarterectomy site was quickly covered by a thin fibrin-platelet coagulum with varying amounts of thrombus. Neoendothelization began before one week and was virtually completed by one month except on areas of heavy thrombus deposition. While this study does not prove the origin of the neoendothelial cell, it suggests that neoendothelial cells are derived from pre-existing endothelium and advance over the endarterectomy site from the junction zone of the normal aorta and to a lesser extent from the orifices of brancing vessels.     

A Preliminary Study on the Effects of Acellular Tissue Graft Augmentation in Acute Achilles Tendon Ruptures

Acute Achilles tendon rupture injuries present surgical challenges because of the mechanical forces placed on this tendon. The purpose of this study was to evaluate the effectiveness of an acellular human dermal tissue matrix, GraftJacket Matrix (Wright Medical Technology, Inc., Arlington, TN), as an augmentation material in acute Achilles tendon repair. Eleven consecutive patients with acute tendon ruptures were evaluated and followed up (20-31 months). Primary repair was followed by augmentation with the graft sutured circumferentially around the tendon. Patients were placed in an early functional rehabilitation program with postoperative evaluation at 3, 6, and 12 months. Outcome scores were calculated based on the American Orthopaedic Foot and Ankle Society ankle-hindfoot scoring system. At 20-month postoperative follow-up, there have been no cases of rerupture or recurrent pain. The average return-to-activity time was 11.8 +/- 0.75 weeks. These retrospective clinical results suggest that with an acellular human dermal tissue matrix to augment acute Achilles tendon, primary repair offers a desirable return-to-activity time without any rerupture or complications. ACFAS Level of Clinical Evidence: 2c.     

Quantification of Massive Allograft Healing with Dynamic Contrast Enhanced-MRI and Cone Beam-CT: A Pilot Study

Although massive allografts are widely used for reconstruction of critical defects in long bones caused by tumor or trauma, many will have inadequate long-term outcomes. Toward a tissue engineering solution to this problem, we developed experimental stem cell and gene therapy adjuvants that induce angiogenesis, osteogenesis, and remodeling of the structural allografts. We present data from pilot studies to show the utility of dynamic contrast enhanced MRI (DCE-MRI) to quantify vascularity after femoral osteotomy in a canine femur model and cone beam CT (CB-CT) to quantify bone volume in a patient after composite prosthetic-allograft reconstructive surgery. The results demonstrate our ability to suppress the artifacts generated by the metal implants required to secure massive allografts such that precise quantification of cortical bone revascularization (>10-fold increase at 3 weeks postoperatively) and new bone formation (accurate to about 193 μm³) around the graft can be performed longitudinally via DCE-MRI and CB-CT, respectively. One or more of the authors have received funding from the Musculoskeletal Transplant Foundation (NE, SK, EMS) and from the NIH (NIH PHS AR054; RNR, EMS). One of the authors (DC) is an employee of Koning Corp, Rochester, NY. Each author certifies that his or her institution has approved the human and animal protocols for this investigation, that all investigations were conducted in conformity with ethical principles of research, and that informed consent for participation in the study was obtained.     

不同植骨材料对跟骨骨折切口愈合的影响

目的研究两种不同植骨材料（白体髂骨、同种异体骨）对跟骨骨折术后切口愈合的影响。方法回顾性研究安徽医科大学第一附属医院自2009年1月至2012年12月采用切开复位AO钢板内固定加植骨术治疗的76例单侧跟骨骨折患者,其中男53例,女23例,平均年龄39．5岁。自体髂骨植骨40例（A组）,同种异体骨植骨36例（B组）。应用统计学方法对患者年龄、伤后至手术时间、切口干燥时间、切口愈合时间、术后24h切口引流量等进行评价。结果A组平均年龄（39．43±11．71）岁,伤后至手术平均时间（10．27±2．54）d,切口平均干燥时间（6．02±1．37）d,切口平均愈合时间（14．08±1．27）d,术后24h平均引流量（78．58±13．92）mL;B组平均年龄（39．53±110．52）岁,伤后至手术平均时间（9．14±3．19）d,切口平均干燥时间（8．08±1．02）d,切口平均愈合时间（16．14±1．84）d,术后24h平均负压引流量（96．69±13．57）mL。A组与B组的切口干燥时间、愈合时间、术后24h引流量的比较差异有统计学意义（P〈0．05）。结论自体髂骨植骨较同种异体骨植骨会缩短跟骨骨折切口的愈合时间。     

A Canine Model of Intimal Hyperplasia (IH) in Autogenous Vein Grafting: A Preliminary Report

High failure rates (10–40% at 1 year and 2–6% per year thereafter) of autologous vein grafts in peripheral bypass surgery due to progressive intimal hyperplasia (IH) have prompted researchers to search for an animal model that develops IH in a relatively short period of time. This study summarizes our experiences in promoting IH in a canine model. Eight to ten centimeters of both jugular veins were exposed in 40 adult mongrel conditioned dogs. After division into 4–5-cm lengths, the segment of jugular vein most proximal was ballooned at 800–900 mm Hg with a modified 8F Fogarty catheter to induce intimal and medial layer injury. The distal segment was left nonballooned. Segments of these autologous vein grafts, 1.5 cm in length, both ballooned and nonballooned, were then anastomosed, end to end, into the carotid and femoral circulations. Six weeks postoperatively the grafts were perfusion-fixed, harvested, and histologically processed, and the amount of the lumen in midgraft sections occupied by IH was determined by image analysis. In all dogs, the degree of IH was significantly greater in the balloon catheterized vs noncatheterized graft segments. IH was more severe in the femoral than in the carotid arteries. The grafts that developed the most severe intimal hyperplasia were femoral grafts that had been balloon catheterized. We conclude that these protocols are effective in inducing IH in a canine model in short postoperative limes.