Alice Harper

Expression of cyclooxygenase-2 in the periprosthetic capsule surrounding a silicone shell implant in the rat.

Prosthetic breast implants are used frequently for both aesthetic and reconstructive purposes. Histologically, the normal tissue response to silicone implants typically involves an inflammatory infiltrate that consists initially of macrophages, and later consists of fibroblasts, myofibroblasts, and lymphocytes. To characterize further the nature of the inflammatory response to silicone breast implants, the authors evaluated the expression of the enzyme cyclooxygenase-2 (COX-2) by the tissues and cells of the breast capsule after silicone augmentation in an animal model. COX-2 is an inducible enzyme that is expressed primarily in response to inflammatory stimuli and mediates the production of prostaglandins that support the inflammatory process. Fifty-nine animals at five time points were evaluated. Specifically, on days 4, 7, 14, 28, and 80 the authors identified cytoplasmic COX-2 expression in each of the three cell types of interest, including endothelial cells, macrophages, and fibroblasts. Selective COX-2 inhibiting agents may be an appropriate pharmacological intervention for modulating the formation of periprosthetic capsules.McLean AL, Talmor M, Harper A, Fahey TJ, Gayle LB, Hoffman LA. Expression of cyclooxygenase-2 in the periprosthetic capsule surrounding a silicone shell implant in the rat.

Development of an acellular bioengineered matrix with a dominant vascular pedicle.

BACKGROUND This study assessed the feasibility of creating a tissue engineering platform by decellularization of fasciocutaneous tissue. MATERIALS AND METHODS A fasciocutaneous flap based upon the superficial inferior epigastric artery was harvested from the abdominal wall of 8-wk-old male Sprague-Dawley rats. All cellular components were removed by sequential treatment with sodium azide, DNAse, and sodium deoxycholate. The degree of decellularization was qualitatively assessed by histology and quantitatively assessed by spectrophotometry. Persistence of relevant extracellular matrix proteins was shown following staining with orcein and hematoxylin. The duration of circuit patency was determined by continuous perfusion with a peristaltic perfusion pump. RESULTS Gross and histologic examination demonstrated removal of cellular constituents with preservation of tissue matrix architecture, including macrochannels and microchannels. This was confirmed by the application of spectrophotometry to DNA isolates, which showed that the decellularized flap retained 4.04 ng/μL DNA, compared with the non-processed control, which retained 37.03 ng/μL DNA, and the acellular control, which was read as having 0.65 ng/μL DNA. The extracellular matrix of vessel walls was shown to remain intact. Peristaltic perfusion of the cannulated pedicle inflow channel with phosphate buffered saline at a rate of 200 μL/min confirmed circuit patency for 6 h. CONCLUSION Fasciocutaneous flaps harvested with an intact vascular pedicle and associated tissue vascular network can be successfully decellularized and perfused ex vivo. This methodology, which is scalable to human size tissues, provides promise as a technique for the production of customizable engineered tissues.

Donor leukocyte migration following extremity transplantation in an experimental model.

In an effort to further define the immunologic mechanisms leading to acute composite-tissue allograft rejection, the migratory patterns of donor leukocytes were evaluated. Using a rat model, 52 orthotopic vascularized hindlimb transplants were performed in strains representing major histocompatibility mismatches. In order to evaluate the effect of allogeneic skin on limb rejection, all donor skin was removed in a second group of allografts. Recipient lymphoid organs were examined during the week following transplantation for antigen-presenting cells using a donor-specific class II monoclonal antibody. Donor leukocytes, with dendritic cell morphology, were identified in recipient spleen and lymph nodes draining the allograft. Significantly higher numbers of donor leukocytes were present during postoperative days 1 through 4 for both groups. Association of these important passenger leukocytes with host T-lymphocytes may represent the site of initiation of the immune response.

Migration of donor leukocytes from limb allografts into host lymphoid tissues.

This study was undertaken in an initial effort to characterize the immunology of extremity transplantation by examining the pattern and kinetics of leukocyte migration from rat limb transplants. Migration of donor leukocytes was evaluated by examining recipient lymphoid tissues with a donor-specific, anti—major histocompatibility complex, class I monoclonal antibody. Double-antibody, two-color labeling was used to localize donor cells to specific regions within these tissues. Donor leukocytes, with dendritic cell morphology, were found in the T-cell-rich areas of lymph nodes draining the allograft and spleen. The donor cells were present on postoperative days 1 through 3 but were not present on days 5 to 7. Donor leukocytes were not present in distant lymph nodes or liver. These findings indicate a migration of leukocytes, most likely the highly immunogenic dendritic cell, from rat limb transplants to the draining lymphoid tissues. Migration occurs shortly after transplantation and may lead to the sensitization of alloreactive T-cells.

COX-2 up-regulation in idiopathic carpal tunnel syndrome

The objective of this study was to determine whether cyclooxygenase-2 (COX-2) is up-regulated in the synovium of patients with carpal tunnel syndrome. Twenty patients were enrolled: 16 consecutive patients with carpal tunnel syndrome and four control patients (exploration for non–carpal tunnel syndrome–related wrist or forearm pathology). Clinical data (demographics, pertinent history, symptomatology) were obtained preoperatively. Flexor tenosynovial tissue was isolated from all patients and clinically graded as thin, intermediate, or thick. Histologic evaluation was conducted to rule out the presence of inflammatory cells. Immunohistochemical staining for COX-2 was performed. The immunohistochemical data were confirmed by reverse transcriptase-polymerase chain reaction analysis of COX-2 mRNA. Results showed that the majority of carpal tunnel syndrome specimens (88 percent) showed synovial hypertrophy compared with 0 percent of the controls (p < 0.05). Also, 69 percent of carpal tunnel syndrome specimens (11 of 16) versus 0 percent of controls (zero of four) stained positively for COX-2 (p < 0.05). Of the carpal tunnel syndrome patients, 91 percent of thick specimens versus 33 percent of intermediate specimens versus 0 percent of thin specimens showed COX-2 staining. The authors conclude that synovial hypertrophy is a prominent finding in carpal tunnel syndrome. COX-2 is up-regulated in the tenosynovium of patients with carpal tunnel syndrome, and this upregulation may correlate with the clinical grade of the tenosynovium. The role of COX-2 in carpal tunnel syndrome may be to mediate remodeling of pathologic tissue. To this end, it may be a potential therapeutic target for specific inhibition.

Optimizing cell sourcing for clinical translation of tissue engineered ears.

Background . Currently, the major impediment to clinical translation of our previously described platform for the fabrication of high fidelity, patient-specific tissue engineered ears is the development of a clinically optimal cell sourcing strategy. A limited autologous auricular chondrocyte (AuC) supply in conjunction with rapid chondrocyte de-differentiation during in vitro expansion currently makes clinical translation more challenging. Mesenchymal stem cells (MSCs) offer significant promise due to their inherent chondrogenic potential, and large availability through minimally invasive procedures. Herein, we demonstrate the promise of AuC/MSC co-culture to fabricate elastic cartilage using 50% fewer AuC than standard approaches. METHODS Bovine auricular chondrocytes (bAuC) and bovine MSC (bMSC) were encapsulated within 10 mg ml-1 type I collagen hydrogels in ratios of bAuC:bMSC 100:0, 50:50, and 0:100 at a density of 25 million cells ml-1 hydrogel. One mm thick collagen sheet gels were fabricated, and thereafter, 8 mm diameter discs were extracted using a biopsy punch. Discs were implanted subcutaneously in the dorsa of nude mice (NU/NU) and harvested after 1 and 3 months. RESULTS Gross analysis of explanted discs revealed bAuC:bMSC co-culture discs maintained their size and shape, and exhibited native auricular cartilage-like elasticity after 1 and 3 months of implantation. Co-culture discs developed into auricular cartilage, with viable chondrocytes within lacunae, copious proteoglycan and elastic fiber deposition, and a distinct perichondrial layer. Biochemical analysis confirmed that co-culture discs deposited critical cartilage molecular components more readily than did both bAuC and bMSC discs after 1 and 3 months, and proteoglycan content significantly increased between 1 and 3 months. CONCLUSION We have successfully demonstrated an innovative cell sourcing strategy that facilitates our efforts to achieve clinical translation of our high fidelity, patient-specific ears for auricular reconstruction utilizing only half of the requisite auricular chondrocytes to fabricate mature elastic cartilage.

Abstract 129: Fabrication of the First Full-Scale Human Auricular Chondrocyte Derived Ear Scaffold for Clinical Application

PURPOSE: Trauma-induced heterotopic ossification (tHO) is the aberrant growth of ectopic bone in soft tissue, which develops in patients following severe musculoskeletal trauma. Much of HO literature focuses on a related pathology known as fibrodysplasia ossificans progressiva (FOP), which is caused by a hyperactivating mutation in the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1 (ACVR1 R206H). Consequently, emphasis has been placed on developing inhibitors with improved specificity for ACVR1. However, patients who develop tHO do not harbor known ACVR1 mutations, and it is unclear whether emphasis on ACVR1-specific inhibition is beneficial for the treatment of tHO. Here investigate whether any single T1-BMPR is required for tHO, or whether these receptors perform overlapping roles during tHO development. We further evaluate the efficacy of the BMP ligand trap, Alk3Fc, as a broad-spectrum inhibitor of T1-BMP receptors in the treatment and prevention tHO.

Abstract: Utilizing a Novel Cell Sourcing Strategy to Fabricate the First Full-Scale Tissue Engineered Human Ear Scaffold

INTRODUCTION: Previously, we fabricated patient-specific auricles using bovine auricular chondrocytes, which displayed effective permanence with structural, biochemical, and mechanical properties similar to native auricular cartilage after 6 months in vivo. However, autologous tissue donation generates a limited cell yield, and further expansion of donor cells can negatively impact chondrogenic capacity. To overcome this challenge, we sought to generate human auricular cartilage through the combined implantation of human auricular chondrocytes (hAuC) with human mesenchymal stem cells (hMSC) as a novel cell sourcing strategy in order to fabricate the first full-scale human ear.

Abstract P1: Bioprinted Vascularized Tissue-Engineered Constructs for In Vivo Perfusion

PurPose: The greatest challenge to contemporary tissue engineers remains the difficulty associated with creating vascular networks within the engineered tissue. Furthermore, any vascularized construct must be designed to allow for anastomosis to the host vascular system. In previous work we synthesized a tissue-engineered scaffold containing an endothelialized internal loop microchannel for microsurgical anastomosis and in vivo perfusion utilizing a sacrificial microfiber technique. Bioprinting is an emerging technology that allows for the consistent and rapid fabrication of three-dimensional constructs comprised of any combination of extrudable polymers and cells with a precise predetermined microarchitecture. Here we describe the fabrication of bioprinted hydrogel constructs for cell seeding and in vivo microanastomosis.

Abstract 197: NOVEL BIODEGRADABLE POLY ESTER-AMIDE (PEA) POLYMER COATING SIGNIFICANTLY REDUCES SUTURE ASSOCIATED INFLAMMATORY RESPONSE

Introduction: Despite their ubiquitous use, surgical sutures are foreign bodies which induce a local immune reaction within the adjacent tissues. This in ammatory reaction predisposes the patient to infection and leads to complications including the development of suture granulomas, tunneling, spitting and abscess formation. We introduce a novel biocompatible and biodegradable (poly (ester-amide) 8-Phe-4 polymer (PEA)) material with anti-in ammatory properties that can be used to coat sutures. We hypothesized that two widely used sutures, plain-gut and silk, would elicit a reduced in ammatory response after being coated with PEA when compared to non-coated controls.