Evaluation of a porcine whole-limb heterotopic autotransplantation model

Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field.

A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.

Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation

BACKGROUND Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. METHODS Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry. RESULTS In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days. CONCLUSIONS We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

Electroporation-mediated interleukin-10 overexpression in skeletal muscle reduces acute rejection in rat cardiac allografts

OBJECTIVES: Human interleukin 10 (hIL-10) may reduce acute rejection after organ transplantation. Our previous data shows that electroporation-mediated transfer of plasmid DNA to peripheral muscle enhances gene transduction dramatically. This study was designed to investigate the effect of electroporation-mediated overexpression of hIL-10 on acute rejection of cardiac allografts in the rat. METHODS: The study was designed to evaluate the effect of hIL-10 gene transfer on (a) early rejection pattern and (b) graft survival. Gene transfer was achieved by intramuscular (i.m.) injection into the tibialis anterior muscle of Fischer (F344) male recipients followed by electroporation 24 h prior to transplantation. Heterotopic cardiac transplantation was performed from male Brown Norway rat to F344. Four groups were studied (n = 6). Treated animals in groups B1 and B2 received 2.5 microg of pCIK hIL-10 and control animals in groups A1 and A2 distilled water. Graft function was assessed by daily palpation. Animals from group A1 were sacrificed at the cessation of the heart beat of the graft and those in group B1 were sacrificed at day 7; blood was taken for ELISA measurement of hIL-10 and tissue for myeloperoxidase (MPO) measurement and histological assessment. To evaluate graft survival, groups A2 and B2 were sacrificed at cessation of the heart beat of the graft. RESULTS: Histological examination revealed severe rejection (IIIB-IV) in group A1 in contrast to low to moderate rejection (IA-IIIA) in group B1 (p = 0.02). MPO activity was significantly lower in group B1 compared to group A1 (18 +/- 7 vs. 32 +/- 14 mU/mg protein, p = 0.05). Serum hIL-10 levels were 46 +/- 13 pg/ml in group B1 vs. 0 pg/ml in group A1. At day 7 all heart allografts in the treated groups B1 and B2 were beating, whereas they stopped beating at 5 +/- 2 days in groups A1 and A2 vs. 14 +/- 2 days in group B2 (p = 0.0012). CONCLUSIONS: Electroporation-mediated intramuscular overexpression of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in rats. This study demonstrates that peripheral overexpression of specific genes in skeletal muscle may reduce acute rejection after whole organ transplantation.

Trochanteric flip osteotomy for cranial extension and muscle protection in acetabular fracture fixation using a Kocher-Langenbeck approach

OBJECTIVE: To describe the advantages and surgical technique of a trochanteric flip osteotomy in combination with a Kocher-Langenbeck approach for the treatment of selected acetabular fractures. DESIGN: Consecutive series, teaching hospital. METHODS: Through mobilization of the vastus lateralis muscle, a slice of the greater trochanter with the attached gluteus medius muscle can be flipped anteriorly. The gluteus minimus muscle can then be easily mobilized, giving free access to the posterosuperior and superior acetabular wall area. Damage to the abductor muscles by vigorous retraction can be avoided, potentially resulting in less ectopic ossification. Ten consecutive cases of acetabular fractures treated with this approach are reported. In eight cases, an anatomic reduction was achieved; in the remaining two cases with severe comminution, the reduction was within one to three millimeters. The trochanteric fragment was fixed with two 3.5-millimeter cortical screws. RESULTS: All osteotomies healed in anatomic position within six to eight weeks postoperatively. Abductor strength was symmetric in eight patients and mildly reduced in two patients. Heterotopic ossification was limited to Brooker classes 1 and 2 without functional impairment at an average follow-up of twenty months. No femoral head necrosis was observed. CONCLUSION: This technique allows better visualization, more accurate reduction, and easier fixation of cranial acetabular fragments. Cranial migration of the greater trochanter after fixation with two screws is unlikely to occur because of the distal pull of the vastus lateralis muscle, balancing the cranial pull of the gluteus medius muscle.

[Endovascular therapy of an abdominal aortic aneurysm in a renal transplant recipient]

INTRODUCTION: The indications for endovascular aortic aneurysm repair (EVAR) are mainly established in hostile abdomen, in patients with significant comorbidities which affect the general operability of the patient and, given the necessary infrastructure, also in ruptured aneurysm. Along to those, we present another possible indication in the presence of a kidney allograft in patients with aortic aneurysm. METHODS: Based on a case report of aorto-biiliac stent-graft repair of an infrarenal aortic aneurysm in a patient with renal allograft, a systematic review of the literature was performed of all similar cases concerning surgical therapy in this constellation. RESULTS: EVAR was performed using an aorto-biiliac system (Zenith) Trifab, COOK) in a 61-year-old male patient 11 years after heterotopic renal allotransplantation in the right iliac fossa. Preoperative renal function was normal. Because the donor renal artery was anastomosed to the recipient's external iliac artery the stent-graft was implanted from the left common femoral artery to minimize temporary allograft ischemia. The intra- and postoperative course was uneventful with a follow-up of presently 12 months. A primary type-II endoleak (retroleak from a lumbar artery) is being treated conservatively so far with embolization being a future option. At present there are 15 cases of EVAR in renal allograft patients that have been reported, all of them being successful. DISCUSSION: All data existing in the literature reported to date as well as our own experience justify the first choice of EVAR in morphologically suitable cases. One major advantage of EVAR in this constellation is the avoidance of aortic cross clamping which poses the graft at risk of ischemia. Long-term results will be most important for definite assessment of EVAR. However, contrast media application during the operation and for CT surveillance should be considered as a major disadvantage.

Femoral head injuries: Which treatment strategy can be recommended?

Despite different operative and non-operative treatment regimens, the outcome after femoral head fractures has changed little over the past decades. The initial trauma itself as well as secondary changes such as posttraumatic osteoarthritis, avascular necrosis or heterotopic ossification is often responsible for severe loss of function of the afflicted hip joint. Anatomic reduction of all fracture fragments seems to be a major influencing factor in determining the outcome quality. Eight years ago we inaugurated a new surgical approach for better access and visualisation for the treatment of femoral head fractures, using the "trochanteric flip" (digastric) osteotomy. Thus inspection of the entire hip joint and accurate fragment reduction under direct visual control are possible. After good initial experiences with this operative procedure we changed our standard treatment regimen to this approach in an attempt to achieve the most accurate anatomic reduction of the femoral head in every affected patient. Between 1998 and 2006 we operated on 12 patients with femoral head fractures associated with posterior hip dislocation, using the new surgical approach. Patients were followed for 2-96 months and outcome was documented with the Merle d'Aubigne and Postel score as well as the Thompson and Epstein score. The posttraumatic formation of heterotopic bone was documented with the Brooker score. Retrospective analysis of these 12 patients showed good or excellent results in 10 patients (83.3%). The two patients with poor outcome developed an avascular necrosis of the femoral head and underwent total hip arthroplasty. Periarticular heterotopic ossification was seen in five patients. In four patients this caused a significantly reduced range of motion and was therefore considered as a posttraumatic complication. The two patients with the most severe heterotopic bone formation (Brooker III and IV) had initially sustained multiple injuries including brain injury. Comparing our results with earlier published series including our own before changing the treatment protocol, the data suggest a favorable outcome in patients with trochanteric flip (digastric) osteotomy for the treatment of femoral head fractures.

Upregulation of endothelin converting enzyme-1 in host liver during chronic cardiac allograft rejection

Endothelin regulates cytokine expression in vitro and in vivo. This study investigated the effects of chronic allograft rejection on hepatic endothelin-converting enzyme-1 (ECE-1) gene expression and endothelin-1 (ET-1) plasma clearance. Using the Lewis-F344 minor histocompatibility mismatch model of heterotopic cardiac transplantation, hepatic ECE-1 gene expression was measured by real-time polymerase chain reaction and host plasma clearance of ET-1 was measured 8 weeks after transplantation in the absence of immunosuppression. In animals undergoing allograft rejection, hepatic ECE-1 gene expression increased 2-fold (P < 0.05), whereas no effect of rejection on ET-1 clearance from plasma was observed. In summary, upregulation of ECE-1 gene expression occurs in the liver of the host during chronic allograft rejection. Because the liver represents both a key organ for cytokine production and for endothelin metabolism, increased hepatic ECE-1-mediated ET-1 synthesis may contribute to host responses and cytokine production during allograft rejection.

Stepped osteotomy of the trochanter for stable, anatomic refixation

Refixation of a trochanteric osteotomy carries a high complication rate. To enhance stability and facilitate anatomic reduction of the trochanteric fragment, we have introduced a stepped osteotomy. Between April 2006 and June 2007, we performed surgical hip dislocations using the modified trochanteric osteotomy combined with a relatively aggressive rehabilitation program. Full weightbearing was allowed at a mean of 42 days (range, 33-54 days). The minimum followup was 8 months (median, 13 months; range, 8-24 months). Postoperative radiographs were assessed prospectively for consolidation or the appearance of malreduction/nonunion/malunion of the osteotomy and heterotopic ossification. In 110 of 113 hips, the trochanteric osteotomy healed in the anatomic position. Two patients had a trochanteric delayed union with loss of anatomic position, and one additional patient underwent revision surgery for a pseudarthrosis and cranial migration of the trochanteric fragment. All three complications related to healing occurred in the first 60 patients when the step height was 3 to 4 mm. After increasing the step heights to 6 mm, we observed no healing complications. Despite more aggressive postoperative mobilization, the incidence of malunion or nonunion related to the new stepped osteotomy is low and approaches zero for steps of 6 mm. It is now our technique of choice.

Die operative Behandlung der Azetabulum-T-Fraktur über eine chirurgische Hüftluxation oder einen Stoppa-Zugang. [Operative treatment of T-type fractures of the acetabulum via surgical hip dislocation or Stoppa approach]

OBJECTIVE: Anatomic reduction and stable fixation by means of tissue- preserving surgical approaches. INDICATIONS Displaced acetabular fractures. Surgical hip dislocation approach with larger displacement of the posterior column in comparison to the anterior column, transtectal fractures, additional intraarticular fragments, marginal impaction. Stoppa approach with larger displacement of the anterior column in comparison to the posterior column. A combined approach might be necessary with difficult reduction. CONTRAINDICATIONS Fractures > 15 days (then ilioinguinal or extended iliofemoral approaches). Suprapubic catheters and abdominal problems (e.g., previous laparotomy due to visceral injuries) with Stoppa approach (then switch to classic ilioinguinal approach). SURGICAL TECHNIQUE: Surgical hip dislocation: lateral decubitus position. Straight lateral incision centered over the greater trochanter. Entering of the Gibson interval. Digastric trochanteric osteotomy with protection of the medial circumflex femoral artery. Opening of the interval between the piriformis and the gluteus minimus muscle. Z-shaped capsulotomy. Dislocation of the femoral head. Reduction and fixation of the posterior column with plate and screws. Fixation of the anterior column with a lag screw in direction of the superior pubic ramus. Stoppa approach: supine position. Incision according to Pfannenstiel. Longitudinal splitting of the anterior portion of the rectus sheet and the rectus abdominis muscle. Blunt dissection of the space of Retzius. Ligation of the corona mortis, if present. Blunt dissection of the quadrilateral plate and the anterior column. Reduction of the anterior column and fixation with a reconstruction plate. Fixation of the posterior column with lag screws. If necessary, the first window of the ilioinguinal approach can be used for reduction and fixation of the posterior column. POSTOPERATIVE MANAGEMENT: During hospital stay, intensive mobilization of the hip joint using a continuous passive motion machine with a maximum flexion of 90 degrees . No active abduction and passive adduction over the body's midline, if a surgical dislocation was performed. Maximum weight bearing 10-15 kg for 8 weeks. Then, first clinical and radiographic follow-up. Deep venous thrombosis prophylaxis for 8 weeks postoperatively. RESULTS: 17 patients with a mean follow-up of 3.2 years. Ten patients were operated via surgical hip dislocation, two patients with a Stoppa approach, and five using a combined or alternative approach. Anatomic reduction was achieved in ten of the twelve patients (83%) without primary total hip arthroplasty. Mean operation time 3.3 h for surgical hip dislocation and 4.2 h for the Stoppa approach. Complications comprised one delayed trochanteric union, one heterotopic ossification, and one loss of reduction. There were no cases of avascular necrosis. In two patients, a total hip arthroplasty was performed due to the development of secondary hip osteoarthritis.

Contractile function is preserved in unloaded hearts despite atrophic remodeling

OBJECTIVE: Recent studies have shown that mechanically unloading a failing heart may induce reverse remodeling and functional improvement. However, these benefits may be balanced by an unloading-related remodeling including myocardial atrophy that might lead to decrease in function. Using a model of heterotopic heart transplantation, we aimed to characterize the myocardial changes induced by long-term unloading. MATERIAL AND METHODS: Macroscopic as well as cellular and functional changes were followed in normal hearts unloaded for a 3-month period. Microscopic parameters were evaluated with stereologic methodology. Myocardial contractile function was quantified with a Langendorff isolated, perfused heart technique. RESULTS: Atrophy was macroscopically obvious and accompanied by a 67% reduction of the myocyte volume and a 43% reduction of the interstitial tissue volume, thus accounting for a shift of the myocyte/connective tissue ratio in favor of noncontractile tissue. The absolute number of cardiomyocyte nuclei decreased from 64.7 +/- 5.1 x 10(7) in controls to 22.6 +/- 3.7 x 10(7) (30 days) and 21.6 +/- 3.1 x 10(7) (90 days) after unloading (P < .05). The numeric nucleic density in the unloaded myocardium, as well as the mean cardiomyocyte volume per cardiomyocyte nucleus, remained constant throughout the 90 days of observation. Functional data indicated an increase in ventricular stiffness, although contractile function was preserved, as confirmed by unaltered maximal developed pressure and increased contractility (maximum rate of left ventricular pressure development) and relaxation (minimum rate of left ventricular pressure development). CONCLUSION: Atrophic remodeling involves both the myocyte and interstitial tissue compartment. These data suggest that although there is decreased myocardial volume and increased stiffness, contractile capacity is preserved in the long-term unloaded heart.

Pre-clinical in vivo models for the screening of bone biomaterials for oral/craniofacial indications: focus on small-animal models.

Preclinical in vivo experimental studies are performed for evaluating proof-of-principle concepts, safety and possible unwanted reactions of candidate bone biomaterials before proceeding to clinical testing. Specifically, models involving small animals have been developed for screening bone biomaterials for their potential to enhance bone formation. No single model can completely recreate the anatomic, physiologic, biomechanic and functional environment of the human mouth and jaws. Relevant aspects regarding physiology, anatomy, dimensions and handling are discussed in this paper to elucidate the advantages and disadvantages of small-animal models. Model selection should be based not on the 'expertise' or capacities of the team, but rather on a scientifically solid rationale, and the animal model selected should reflect the question for which an answer is sought. The rationale for using heterotopic or orthotopic testing sites, and intraosseous, periosseous or extraskeletal defect models, is discussed. The paper also discusses the relevance of critical size defect modeling, with focus on calvarial defects in rodents. In addition, the rabbit sinus model and the capsule model in the rat mandible are presented and discussed in detail. All animal experiments should be designed with care and include sample-size and study-power calculations, thus allowing generation of meaningful data. Moreover, animal experiments are subject to ethical approval by the relevant authority. All procedures and the postoperative handling and care, including postoperative analgesics, should follow best practice.