Soluble allogenic telocollagen as a direct protein therapeutic: Results of serial injections in a rodent rotator cuff tear model.

HYPOTHESIS: Delivery of soluble allogeneic type I telocollagen (allo-telocollagen) will accelerate and improve the healing of damaged tendons. Our hypothesis draws from known mechanochemical properties of type I collagen that direct its incorporation into damaged connective tissue. We further suggest that allo-telocollagen will raise a minimal immunogenic reaction due to homology within species. METHODS: Seventy-eight shoulders (39 Sprague-Dawley rats) had their supraspinatus tendon surgically detached from its footprint on the humerus and repaired (72 shoulders) or left uninjured (6 shoulders). The repaired tissue was treated with an injection of 100 µl of saline, 10 mg/ml allogeneic atelocollagen (allo-atelocollagen), or 10 mg/ml allo-telocollagen at 0-, 1-, and 2-weeks post-surgery. At 30- and 60-days post-surgery, the tendons were assessed by mechanical testing (failure load, failure stress, stiffness, and relaxation) and by semiquantitative histological scoring. RESULTS: At 30-days post-surgery, the mechanical and histological outcomes were not statistically different. However, at day 60, allo-telocollagen improved the failure strength of the supraspinatus (29.9 ± 4.7 N) relative to saline (20.0 ± 3.5 N; P value <= 0.001) or allo-atelocollagen (23.2 ± 1.5 N; P value = 0.025) treated tendons, and it approached that of uninjured controls (36.9 ± 5.0 N; P value = 0.021). Allo-telocollagen improved the failure stress of the supraspinatus (34.1 ± 9.3 MPa) relative to the saline treated tendons (21.4 ± 6.0 MPa; P value = 0.031; 160% improvement) and was no different than uninjured controls (33.4 ± 9.9 MPa; P value = 0.999) or allo-atelocollagen (32.3 ± 7.4 MPa; P value = 0.977). The stiffness of uninjured controls was far greater than any of injured/treated tendons (>200% stiffer). Histological scoring showed that the allo-telocollagen treated tendons produced better collagen fiber arrangement (1.55 ± 0.17) than saline (2.50 ± 0.29; P value = 0.001) or allo-atelocollagen (2.23 ± 0.28; P value = 0.042) treated tendons and that it did not increase markers of immunogenesis (1.10 ± 0.42) relative to either saline (1.44 ± 0.20; P value = 0.369) or allo-atelocollagen (0.68 ± 0.41; P value = 0.1058). CONCLUSIONS: While all three treatments produced similar results at 30 days, by 60 days, soluble allo-telocollagen clearly separated from the other interventions, yielding better mechanical and histological outcomes in a torn/repaired rotator cuff rat model. Allo-telocollagen treated tendons also approached the failure strength and matched the failure stresses of uninjured control tendons. The data suggest a new use for allo-telocollagen as a deliverable direct protein mechanotherapeutic that can improve both healing quality and speed.

Photosealed Neurorrhaphy Using Autologous Tissue.

Photochemical sealing of a nerve wrap over the repair site isolates and optimizes the regenerating nerve microenvironment. To facilitate clinical adoption of the technology, we investigated photosealed autologous tissue in a rodent sciatic nerve transection and repair model. Rats underwent transection of the sciatic nerve with repair performed in three groups: standard microsurgical neurorrhaphy (SN) and photochemical sealing with a crosslinked human amnion (xHAM) or autologous vein. Functional recovery was assessed at four-week intervals using footprint analysis. Gastrocnemius muscle mass preservation, histology, and nerve histomorphometry were evaluated at 120 days. Nerves treated with a PTB-sealed autologous vein improved functional recovery at 120 days although the comparison between groups was not significantly different (SN: -58.4 +/- 10.9; XHAM: -57.9 +/- 8.7; Vein: -52.4 +/- 17.1). Good muscle mass preservation was observed in all groups, with no statistical differences between groups (SN: 69 +/- 7%; XHAM: 70 +/- 7%; Vein: 70 +/- 7%). Histomorphometry showed good axonal regeneration in all repair techniques. These results demonstrate that peripheral nerve repair using photosealed autologous veins produced regeneration at least equivalent to current gold-standard microsurgery. The use of autologous veins removes costs and foreign body concerns and would be readily available during surgery. This study illustrates a new repair method that could restore normal endoneurial homeostasis with minimal trauma following severe nerve injury.

Continuous oxygen monitoring to enhance ex-vivo organ machine perfusion and reconstructive surgery.

Continuous oxygenation monitoring of machine-perfused organs or transposed autologous tissue is not currently implemented in clinical practice. Oxygenation is a critical parameter that could be used to verify tissue viability and guide corrective interventions, such as perfusion machine parameters or surgical revision. This work presents an innovative technology based on oxygen-sensitive, phosphorescent metalloporphyrin allowing continuous and non-invasive oxygen monitoring of ex-vivo perfused vascularized fasciocutaneous flaps. The method comprises a small, low-energy optical transcutaneous oxygen sensor applied on the flap's skin paddle as well as oxygen sensing devices placed into the tubing. An intermittent perfusion setting was designed to study the response time and accuracy of this technology over a total of 54 perfusion cycles. We further evaluated correlation between the continuous oxygen measurements and gold-standard perfusion viability metrics such as vascular resistance, with good agreement suggesting potential to monitor graft viability at high frequency, opening the possibility to employ feedback control algorithms in the future. This proof-of-concept study opens a range of research and clinical applications in reconstructive surgery and transplantation at a time when perfusion machines undergo rapid clinical adoption with potential to improve outcomes across a variety of surgical procedures and dramatically increase access to transplant medicine.

Gal - T Knockout Porcine Nerve Xenografts Support Axonal Regeneration in a Rodent Sciatic Nerve Model.

BACKGROUND: Nerve xenografts harvested from transgenic α1,3-galactosyltransferase knockout (GalT-KO) pigs lack the epitope responsible for hyperacute rejection in pig-to-primate transplants. It is unknown whether these cold preserved nerve grafts support axonal regeneration in another species during and after immunosuppression. In this study, we compare outcomes between autografts and cold preserved xenografts in a rat sciatic model of nerve gap repair. METHODS: Fifty male Lewis rats had a 1 cm sciatic nerve defect repaired using either: autograft and suture (n=10); 1-week or 4-week cold preserved xenograft and suture (n=10 per group); 1-week or 4-week cold preserved xenograft and photochemical tissue bonding using a human amnion wrap (PTB/HAM) (n=10 per group). Rats with xenografts were given tacrolimus until 4 months post-operatively. At 4 and 7 months, rats were euthanized and nerve sections harvested. Monthly sciatic functional index (SFI) scores were calculated. RESULTS: All groups showed increases in SFI scores by 4 and 7 months. The autograft suture group had the highest axon density at 4 and 7 months. The largest decrease in axon density from 4 to 7 months was in the 1-week cold preserved PTB/HAM group. The only significant difference between group SFI scores occurred at 5 months, when both 1-week cold preserved groups had significantly lower scores than the 4-week cold preserved suture group. CONCLUSIONS: Our results in the rat sciatic model suggest that GalT-KO nerve xenografts may be viable alternatives to autografts and demonstrate the need for further studies of long-gap repair and comparison with acellular nerve allografts. CLINICAL RELEVANCE: This proof-of-concept study in the rat sciatic model demonstrates that cold preserved GalT-KO porcine xenografts support axonal regeneration, as well as axonal viability following immunosuppression withdrawal. These results further suggest a role for both cold preservation and photochemical tissue bonding in modulating the immunological response at the nerve repair site.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

The efficacy of antibiotic-eluting material in a two-stage model of periprosthetic joint infection.

Periprosthetic joint infections occur in about 2% of patients who undergo primary total joint arthroplasty, a procedure performed over 1 million times in the United States. The gold standard of treatment is a two-stage revision. This study aimed to establish a two-stage procedure in a preclinical small animal model (rat) to test and compare the efficacy of an antibiotic-eluting material in managing infection. Joint replacement was simulated by transchondylarly implanting a polyethylene (PE) plug into the distal femur and a titanium screw in the proximal tibia. Methicillin-sensitive Staphylococcus aureus (MSSA) 108 CFU/mL was injected into the tibial canal and the joint space before wound closure. The control groups were killed on postoperative day (POD) 18 (n = 12) and on POD 42 (n = 4) to assess both early and later-stage outcomes in the control group. The test group underwent revision surgery on POD 18 for treatment using gentamicin-eluting polyethylene (GPE, n = 4) and was observed until POD 42 to evaluate the efficacy of treatment. Our results showed that the bone loss for the treatment group receiving GPE was significantly less than that of the control (p < 0.05), which was supported by the histology images and an AI-tool assisted infection rate evaluation. Gait metrics duty factor imbalance and hindlimb temporal symmetry were significantly different between the treatment and control groups on Day 42. This animal model was feasible for evaluating treatments for peri-prosthetic joint infections (PJI) with a revision surgery and specifically that revision surgery and local antibiotic treatment largely hindered the peri-prosthetic bone loss. Statement of clinical significance: This revision model of peri-prosthetic infection has the potential of comparatively evaluating prophylaxis and treatment strategies and devices. Antibiotic-eluting UHMWPE is devised as at tool in treating PJI while providing weight bearing and joint space preservation.

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study.

Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques.

A longitudinal rat model for assessing postoperative recovery and bone healing following tibial osteotomy and plate fixation.

BACKGROUND: Rodent models are commonly employed to validate preclinical disease models through the evaluation of postoperative behavior and allodynia. Our study investigates the dynamic interplay between pain and functional recovery in the context of traumatic osteotomy and surgical repair. Specifically, we established a rat model of tibial osteotomy, followed by internal fixation using a 5-hole Y-plate with 4 screws, to explore the hypothesis that histological bone healing is closely associated with functional recovery. OBJECTIVE: Our primary objective was to assess the correlation between bone healing and functional outcomes in a rat model of tibial osteotomy and plate fixation. METHODS: Seventeen male Sprague-Dawley rats underwent a metaphyseal transverse osteotomy of the proximal tibia, simulating a fracture-like injury. The resultant bone defect was meticulously repaired by realigning and stabilizing the bone surfaces with the Y-plate. To comprehensively assess recovery and healing, we performed quantitative and qualitative evaluations at 2, 4, 6, and 8 weeks post-surgery. Evaluation methods included micro-CT imaging, X-ray analysis, and histological examination to monitor bone defect healing. Concurrently, we employed video recording and gait analysis to evaluate functional recovery, encompassing parameters such as temporal symmetry, hindlimb duty factor imbalance, phase dispersion, and toe spread. RESULTS: Our findings revealed complete healing of the bone defect at 8 weeks, as confirmed by micro-CT and histological assessments. Specifically, micro-CT data showed a decline in fracture volume over time, indicating progressive healing. Histological examination demonstrated the formation of new trabecular bone and the resolution of inflammation. Importantly, specific gait analysis parameters exhibited longitudinal changes consistent with bone healing. Hindlimb duty factor imbalance, hindlimb temporal symmetry, and phase dispersion correlated strongly with the healing process, emphasizing the direct link between bone healing and functional outcomes. CONCLUSIONS: The establishment of this tibia osteotomy model underscores the association between bone healing and functional outcomes, emphasizing the feasibility of monitoring postoperative recovery using endpoint measurements. Our overarching objective is to employ this model for assessing the local efficacy of drug delivery devices in ameliorating post-surgical pain and enhancing functional recovery.

Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo.

The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair.

The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression.

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

Hydrogel device for analgesic drugs with in-situ loading and polymerization.

The high prevalence of opioid addiction and the shortcomings of systemic opioids has increased the pace of the search for alternative methods of pain management. The local delivery of pain medications has started to be used as a tool for pain management and to decrease the use of systemic opioids for these patients. Here, we explored an in-situ polymerizable hydrogel system for the local delivery of analgesics and nonsteroid anti-inflammatory drugs (NSAID) for orthopaedic applications. We synthesized a series of methacrylated oligomeric polyethylene glycol-co-lactic acid polymer using microwave radiation for the delivery of bupivacaine hydrochloride as an analgesic and ketorolac tromethamine as an NSAID. We determined drug elution and gel degradation profiles in vitro. Biocompatibility was assessed against osteoblasts in vitro and by histological analysis after subcutaneous implantation for 4 weeks in vivo. Intra-articular and systemic concentrations and pharmacokinetic parameters were estimated using a two-compartment pharmacodynamic model based on in-vitro elution profiles. This type of in-situ applicable hydrogels is promising for extending the local efficacy of pain medication and further reducing the need for opioids.

Bone Marrow Stem Cells with Tissue-Engineered Scaffolds for Large Bone Segmental Defects: A Systematic Review.

Critical-sized bone defects (CSBDs) represent a significant clinical challenge, stimulating researchers to seek new methods for successful bone reconstruction. The aim of this systematic review is to assess whether bone marrow stem cells (BMSCs) combined with tissue-engineered scaffolds have demonstrated improved bone regeneration in the treatment of CSBD in large preclinical animal models. A search of electronic databases (PubMed, Embase, Web of Science, and Cochrane Library) focused on in vivo large animal studies identified 10 articles according to the following inclusion criteria: (1) in vivo large animal models with segmental bone defects; (2) treatment with tissue-engineered scaffolds combined with BMSCs; (3) the presence of a control group; and (4) a minimum of a histological analysis outcome. Animal research: reporting of in Vivo Experiments guidelines were used for quality assessment, and Systematic Review Center for Laboratory animal Experimentation's risk of bias tool was used to define internal validity. The results demonstrated that tissue-engineered scaffolds, either from autografts or allografts, when combined with BMSCs provide improved bone mineralization and bone formation, including a critical role in the remodeling phase of bone healing. BMSC-seeded scaffolds showed improved biomechanical properties and microarchitecture properties of the regenerated bone when compared with untreated and scaffold-alone groups. This review highlights the efficacy of tissue engineering strategies for the repair of extensive bone defects in preclinical large-animal models. In particular, the use of mesenchymal stem cells, combined with bioscaffolds, seems to be a successful method in comparison to cell-free scaffolds.

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

Acellular Nipple Scaffold Development, Characterization, and Preliminary Biocompatibility Assessment in a Swine Model.

BACKGROUND: The standard in nipple reconstruction remains the autologous skin flap. Unfortunately, the results are not satisfying, with up to 75% loss of nipple projection over time. Existing studies investigated the use of primates as a source of implants. The authors hypothesized that the porcine nipple can serve as a perfect shape-supporting implant because of functional similarities to the human nipple. A decellularization protocol was developed to obtain an acellular nipple scaffold (ANS) for nipple reconstruction. METHODS: Tissue samples were collected from eight disease-free female Yorkshire pigs (60 to 70 kg) and then decellularized. The decellularization efficiency and extracellular matrix characterization was performed histologically and quantitatively (DNA, total collagen, elastin, and glycosaminoglycan content). In vitro and in vivo biocompatibility was determined by human dermal fibroblast culture and subcutaneous implantation of six ANSs in a single Yorkshire pig (60 to 70 kg), respectively. Inflammation and adverse events were monitored daily based on local clinical signs. RESULTS: The authors showed that all cellular structures and 96% of DNA [321.7 ± 57.6 ng DNA/mg wet tissue versus 11.7 ± 10.9 ng DNA/mg wet tissue, in native and ANS, respectively ( P < 0.001)] can be successfully removed. However, this was associated with a decrease in collagen [89.0 ± 11.4 and 58.8 ± 9.6 µg collagen/mg ( P < 0.001)] and elastin [14.2 ± 1.6 and 7.9 ± 2.4 µg elastin/mg ( P < 0.05)] and an increase in glycosaminoglycan content [5.0 ± 0.7 and 6.0 ± 0.8 ng/mg ( P < 0.05)]. ANS can support continuous cell growth in vitro and during preliminary biocompatibility tests in vivo. CONCLUSION: This is a preliminary report of a novel promising ANS for nipple reconstruction, but more research is needed to validate results. CLINICAL RELEVANCE STATEMENT: Breast cancer is very common among women. Treatment involves mastectomy, but its consequences affect patient mental well-being, and can lead to depression. Nipple-areola complex reconstruction is critical, and existing methods lead to unsatisfactory outcomes.

Photo-crosslinked GelMA loaded with dental pulp stem cells and VEGF to repair critical-sized soft tissue defects in rats.

BACKGROUND: Tissue engineering of skin and mucosa is essential for the esthetic and functional reconstruction of individuals disfigured by trauma, resection surgery, or severe burns while overcoming the limited amount of autograft and donor site morbidity. PURPOSE: We aimed to determine whether a combination of Gelatin-methacryloyl (GelMA) hydrogel scaffold alone or loaded with either dental pulp stem cells (DPSCs) and/or vascular endothelial growth factor (VEGF) could improve skin wound healing in rats. MATERIALS AND METHODS: Four 10 mm full-thickness skin defects were created on the dorsum of 15 Sprague-Dawley rats. The wounds were treated with GelMA alone, GelMA+DPSCs, or GelMA+DPSCs+VEGF. Unprotected wounds were used as controls. Animals were euthanized at 1-, 2-, and 4 weeks post-surgery, and the healing wounds were harvested for clinical, histological, and RT-PCR analysis. RESULTS: No signs of clinical inflammation were observed among all groups. Few and sparse mononuclear inflammatory cells were observed in GelMA+DPSCs and GelMA+DPSCs+VEGF groups at 2 weeks, with complete epithelialization of the wounds. At 4 weeks, the epidermis in GelMA+DPSCs and GelMA+DPSCs+VEGF groups was indistinguishable from the empty defect and GelMA groups. The decrease in cellularity and increase in density of collagen fibers were observed over time in both GelMA+DPSCs and GelMA+DPSCs+VEGF groups but were more evident in the GelMA+DPSCs+VEGF group. The GelMA+DPSCs+VEGF group showed a higher expression of the KER 10 gene at all time points compared with the other groups. Expression of Col1 A1 and TGFß-1 were not statistically different over time neither among the groups. CONCLUSION: GelMA scaffolds loaded with DPSCs, and VEGF accelerated the re-epithelialization of skin wounds.

Light-Activated Vascular Anastomosis.

Background. There have been few advances in technique since vascular anastomosis was performed with silk suture on a curved needle in 1902. This technique results in disruption of the endothelium with exposed intraluminal suture, both of which may lead to thrombocyte aggregation, intimal hyperplasia, and vascular stenosis. A variety of alternative techniques have been explored, with limited success. Photochemical tissue bonding (PTB) is a light-activated methodology of rapidly cross-linking tissue interfaces at the molecular level. Herein, we describe a new technique for anastomosis of venous interposition graft in an ovine model of femoral artery bypass utilizing PTB. Methods. Polypay specific pathogen free sheep (n = 5; 40-45 kg) underwent femoral artery bypass utilizing saphenous vein. The femoral artery was transected and reversed saphenous vein was implanted as an interposition graft. The proximal anastomosis was created as a vein-over-artery cuff utilizing PTB, and the distal anastomosis was created with standard interrupted 8-0 polypropylene suture. Four weeks post-index operation, femoral angiogram was performed to evaluate patency, tortuosity, and luminal diameter. All bypass grafts were harvested and longitudinal and transverse histological sections from the proximal anastomosis were analyzed. Results. The PTB anastomoses (n = 5) were immediately watertight and patent. All animals survived the 28-day study duration. Angiography revealed patent grafts with no aneurysm or stenosis (n = 5). Histologic examination revealed integration of the venous endothelium with the arterial adventitia. Conclusion. Photochemical tissue bonding creates an immediate strong, watertight vascular anastomosis that can withstand physiologic arterial pressure and remains patent at 28 days without the need for intraluminal suture.

Tolerance of a Vascularized Composite Allograft Achieved in MHC Class-I-mismatch Swine via Mixed Chimerism.

Background: Vascularized composite allografts (VCAs) allow reconstruction of devastating injuries and amputations, yet require lifelong immunosuppression that is associated with significant morbidity. Induction of immune tolerance of VCAs would permit widespread use of these procedures. VCAs are acquired from deceased donors most likely to be fully-MHC-mismatched (in contrast to living-related renal transplant donor-recipient pairs matched at one MHC haplotype). After achieving VCA tolerance in a swine model equivalent to clinical living-related renal transplants (single-haplotype MHC mismatches: e.g., "mother-daughter"/haploidentical), we tested our protocol in MHC class I, class II, and fully-MHC-mismatched pairs. Although class II mismatched swine demonstrated similar results as the haploidentical scenario (stable mixed chimerism and tolerance), our protocol failed to prevent rejection of class I and full mismatch VCAs. Here, we describe a new adapted conditioning protocol that successfully achieved tolerance across MHC class-I-mismatch barriers in swine. Methods: Swine were treated with non-myeloablative total body and thymic irradiation two days prior to infusion of bone marrow cells from an MHC class I-mismatched donor. They also received a short-term treatment with CTLA4-Ig (Belatacept®) and anti-IL6R mAb (Tociluzimab®) and were transplanted with an osteomyocutaneous VCA from the same donor. Results: Stable mixed chimerism and tolerance of MHC class-I-mismatched VCAs was achieved in 3 recipients. Allograft tolerance was associated with a sustained lack of anti-donor T cell response and a concomitant expansion of double negative CD4-CD8- T cells producing IL-10. Conclusions: This study demonstrates the first successful mixed chimerism-induced VCA tolerance in a large animal model across a MHC class-I-mismatch. Future studies aimed at fully-mismatched donor-recipient pairs are under investigation with this protocol.

A Photosealed Cap Prevents Disorganized Axonal Regeneration and Neuroma following Nerve Transection in Rats.

Neuroma is a common sequela of traumatic peripheral nerve injury that can result in pain and decreased quality of life for patients. Neuromas result from axonal outgrowth in an attempt to reestablish continuity with the disrupted distal nerve end. Photosealing is a light-activated technique whereby tissues can be securely isolated in a strong and secure manner. This study investigated whether photosealing of autologous vein and crosslinked human amniotic membrane (xHAM) to cap the proximal stump of transected sciatic nerve would prevent disorganized axonal regeneration and neuroma in a rat model. Methods: The right sciatic nerve of Lewis rats (n = 27, 300-350 g) was transected 1 cm proximal to the trifurcation. Animals were randomized to one of three groups (n = 9): no further intervention (Group 1), photosealing with xHAM (Group 2), or photosealing with vein (Group 3). After 60 days, rats were euthanized and their right hindlimbs were re-explored for evidence of disorganized axonal regeneration and/or bulbous neuroma. Results: All untreated control animals were found to have protruding nerve fibers, often invading the adjacent muscle, and 33% of these control animals exhibited a bulbous neuroma. Photosealing with xHAM successfully capped 100% of nerves, with no observable axonal outgrowth. Photosealing with vein prevented axonal outgrowth in eight of nine nerves. No bulbous neuroma was found in any photosealed nerves. Conclusion: Nerve capping with photosealed xHAM or autologous vein can prevent axonal outgrowth in transected nerves, therefore decreasing the likelihood of symptomatic neuroma formation following nerve transection injury or surgical intervention.

Light-activated photosealing with human amniotic membrane strengthens bowel anastomosis in a hypotensive, trauma-relevant swine model.

BACKGROUND: Gastrointestinal anastomotic leakage is a dreaded complication despite advancements in surgical technique. Photochemical tissue bonding (PTB) is a method of sealing tissue surfaces utilizing photoactive dye. We evaluated if crosslinked human amniotic membrane (xHAM) photosealed over the enteroenterostomy would augment anastomotic strength in a trauma-relevant swine hemorrhagic shock model. METHODS: Yorkshire swine (40-45 kg, n = 14) underwent midline laparotomy and sharp transection of the small intestine 120 cm proximal to the ileocecal fold. Immediately following intestinal transection, a controlled arterial bleed was performed to reach hemorrhagic shock. Intestinal repair was performed after 60 minutes and autotransfusion of the withdrawn blood was performed for resuscitation. Animals were randomized to small intestinal anastomosis by one of the following methods (seven per group): suture repair (SR), or SR with PTB augmentation. Animals were euthanized at postoperative Day 28 and burst pressure (BP) strength testing was performed on all excised specimens. RESULTS: Mean BP for SR, PTB, and native tissue groups were 229 ± 40, 282 ± 21, and 282 ± 47 mmHg, respectively, with the SR group statistically significantly different on analysis of variance (p = 0.02). Post-hoc Tukey all-pairs comparison demonstrated a statistically significant difference in burst pressure strength between the SR only and the PTB group (p = 0.04). All specimens in SR group ruptured at the anastomosis upon burst pressure testing, while all specimens in the PTB group ruptured at least 2.5 cm from the anastomosis. CONCLUSION: Photosealing with xHAM significantly augments the strength of small intestinal anastomosis performed in a trauma porcine model.

Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts.

BACKGROUND: Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. METHODS: In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). RESULTS: During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). CONCLUSIONS: Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS.