Yvonne I. Heit

External volume expansion increases subcutaneous thickness, cell proliferation, and vascular remodeling in a murine model.

Background: Fat grafting is a powerful tool for soft-tissue reconstruction; however, the science behind recipient bed preparation has not been thoroughly explored. External volume expansion using suction before fat grafting has been used clinically to improve reliability and consistency of graft survival. The authors developed a murine model to investigate the underlying mechanism of external volume expansion. Methods: The authors created an external volume expansion device using a soft-silicone dome connected to a vacuum source (25 mmHg) to treat the dorsum of mice, and the response was compared with treatment with an occlusive dressing. Treated areas were monitored with magnetic resonance imaging. Remodeling of microvasculature was studied with corrosion casting on day 7. Effects on tissue thickness, number of adipocytes, cell proliferation, and blood vessel density were analyzed at 28 days. Results: Macroscopic analysis showed tissue swelling at sites treated with the external volume expansion device by 21 days, without skin damage. On day 28, external volume expansion increased the thickness of the subcutaneous fat layer twofold, consistent with magnetic resonance imaging observations. The proliferation rate in the subcutaneous layer of expansion-treated areas increased twofold, with a net 2.2-fold increase in number of adipocytes in columns; remodeling of the vessels network occurred, with reorientation and increase of vessel diameters shown by corrosion casting and 1.9-fold augmentation of vessels density. Conclusions: External volume expansion applied to mouse integument induces highly proliferative and vascularized subcutaneous tissue. Recipient-site preparation using external volume expansion devices may be a promising tool to enhance cell and tissue engraftment.

Mast cells are required in the proliferation and remodeling phases of microdeformational wound therapy.

Background: Mast cells are important in numerous inflammatory processes. They are also mechanosensitive and likely play an important role in wound healing. The authors hypothesized that mechanical alteration of the wound environment with a distributed suction device could link mast cells to the healing cascade. Methods: Controlled uniform full-thickness wound surface microdeformations were induced by suction combined with an open-pore polyurethane foam (microdeformational wound therapy) in mast cell–deficient WWv mice and their mast cell–sufficient littermates. Wound healing parameters were assessed in the inflammatory, proliferative, and remodeling phases of healing. Results: Wound tissue granulation, cell proliferation, blood vessel sprouting, and collagen maturation were found to be mast cell–dependent throughout the proliferating and remodeling stages of healing. Conclusion: Mast cells are critical in the robust granulation tissue response seen in microdeformational wound therapy and in the modulation of the remodeling phase of wound healing.

Nonactivated versus thrombin-activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro.

Background: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing. Methods: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control. Results: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets. Conclusions: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered “on demand” rather than in an initial bolus.

Mesenchymal Stromal Cell Mutations and Wound Healing Contribute to the Etiology of Desmoid Tumors

Desmoid tumors are nonmalignant neoplasms of mesenchymal origin that mainly contain fibroblast lineage cells. These tumors often occur in patients with familial adenomatous polyposis (FAP) coli who have germ line mutations in the APC gene. Given emerging data that has implicated multipotent mesencyhmal stromal cells (MSC) in the origin of mesenchymal tumors, we hypothesized that desmoid tumors may arise in patients with FAP after MSCs acquire somatic mutations during the proliferative phase of wound healing. To test this idea, we examined 16 desmoid tumors from FAP-associated and sporadic cases, finding that all 16 of 16 tumors expressed stem cell markers, whereas matching normal stromal tissues were uniformly negative. Desmoid tumors also contained a subclass of fibrocytes linked to wound healing, angiogenesis, and fibrosis. Using an MSC cell line derived from an FAP-associated desmoid tumor, we confirmed an expected loss in the expression of adenomatous polyposis coli (APC) and the transcriptional repressor BMI-1 while documenting the coexpression of markers for chondrocytes, adipocytes, and osteocytes. Together, our findings argue that desmoid tumors result from the growth of MSCs in a wound healing setting that is associated with deregulated Wnt signaling due to APC loss. The differentiation potential of these MSCs combined with expression of BMI-1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggests that desmoid tumors may respond to therapies targeting these pathways.

Foam pore size is a critical interface parameter of suction-based wound healing devices.

Background: Suction-based wound healing devices with open-pore foam interfaces are widely used to treat complex tissue defects. The impact of changes in physicochemical parameters of the wound interfaces has not been investigated. Methods: Full-thickness wounds in diabetic mice were treated with occlusive dressing or a suction device with a polyurethane foam interface varying in mean pore size diameter. Wound surface deformation on day 2 was measured on fixed tissues. Histologic cross-sections were analyzed for granulation tissue thickness (hematoxylin and eosin), myofibroblast density (&agr;-smooth muscle actin), blood vessel density (platelet endothelial cell adhesion molecule-1), and cell proliferation (Ki67) on day 7. Results: Polyurethane foam–induced wound surface deformation increased with polyurethane foam pore diameter: 15 percent (small pore size), 60 percent (medium pore size), and 150 percent (large pore size). The extent of wound strain correlated with granulation tissue thickness that increased 1.7-fold in small pore size foam–treated wounds, 2.5-fold in medium pore size foam–treated wounds, and 4.9-fold in large pore size foam–treated wounds (p < 0.05) compared with wounds treated with an occlusive dressing. All polyurethane foams increased the number of myofibroblasts over occlusive dressing, with maximal presence in large pore size foam–treated wounds compared with all other groups (p < 0.05). Conclusions: The pore size of the interface material of suction devices has a significant impact on the wound healing response. Larger pores increased wound surface strain, tissue growth, and transformation of contractile cells. Modification of the pore size is a powerful approach for meeting biological needs of specific wounds.

Early kinetics of integration of collagen-glycosaminoglycan regenerative scaffolds in a diabetic mouse model.

Background: Collagen-glycosaminoglycan scaffolds, originally designed to treat severe burns, are now commonly used in patients with complex wounds associated with diabetes mellitus. In this study, the authors investigated how the thickness of the scaffold would affect cellular integration with the diabetic host and whether this can be accelerated using subatmospheric pressure wound therapy devices. Methods: Collagen-glycosaminoglycan scaffolds, 500 to 2000-&mgr;m thick, were applied to dorsal wounds in genetically diabetic mice. In addition, 1000-&mgr;m collagen-glycosaminoglycan scaffolds with and without silicone were treated with a subatmospheric pressure device (−125 mmHg). On days 5 and 10, cellular and vascular integration of tissues was studied by histology, immunohistochemistry, corrosion casting, and qRT-polymerase chain reaction. Results: Cells and vessels from the wound surface populated the scaffold to form layers with varying cellular density. Areas of high cell density and proliferation were noted at the bottom of the scaffold. Increasing the thickness of the scaffold did not affect the extent of cellular ingrowth, so that thicker scaffolds had a thicker residual acellular layer on the surface. The thickness of cellular ingrowth was stable between days 5 and 10, whereas vessels seen in the scaffolds on day 10 were not yet present on day 5. Subatmospheric pressure devices applied to silicone-covered collagen-glycosaminoglycan scaffolds minimized the granulation tissue formation beneath the scaffold, which enhanced vessel ingrowth. Conclusions: The early kinetics of cellular integration into collagen-glycosaminoglycan scaffolds is independent of scaffold thickness in a diabetic wound model. Scaffold adherence to the wound and integration can be improved using a subatmospheric pressure device.

Abstract 484: Persistent activation of mesenchymal stromal cells (MSCs) and tissue repair in the etiology of desmoid tumors (DT)

Mesenchymal stromal cells (MSCs) are multipotent cells that can be isolated from various adult tissues and are implicated in a growing list of sarcomas. Desmoid tumors (DTs) have a clonal origin, are linked with wound healing, and mostly arise in the abdomen of patients with familial adenomatous polyposis (FAP) or sporadically. MSCs home to sites of tissue damage and the persistent activation of these cells in a wound healing context combined with deregulated Wnt signaling may lead to neoplasia. To test this idea, we performed immunohistochemistry on 17 sporadic and FAP-associated DTs from different individuals for the expression of multiple MSC markers (CD73, CD90, and Bmi1). While present in differing numbers, all specimens showed positive cells for all markers, and immunofluorescence (IF) showed co-localization of these markers and CD105 in cells within tumors. Matching adjacent normal fascia from a subset was negative for these antigens. A FAP DT-derived cell line was then made that also co-expressed these MSC markers but not CD34, a negative marker. Fluorescence-activated cell sorting analysis of surface antigen profiles confirmed that >90% of the cell population bore MSC positive, but not negative, markers. Although not genotyped, IF for the C- vs. N-termini of APC confirmed that only truncated APC product was expressed in our DT-derived MSC line. Upon growth for 5-6 weeks in differentiation medium, we confirmed by co-expression of CTGF and Sox9 (obligate chondrocyte markers) plus Alcian blue staining, PPARγ and c/EBPα (obligate adipocyte markers) plus Oil Red staining, and OPN and Runx2 (obligate osteocyte markers) plus von Kossa staining that the DT-derived MSCs were tripotent. In conclusion, this study supports the view that DT etiology involves the persistence of MSCs with deregulated Wnt signaling. The differentiation potential of these cells combined with expression of Bmi1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggest that inhibitors targeting these pathways may provide a rational systemic therapy to prevent primary or recurrent DT in high-risk patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2011-484

LOP32: Early kinetics of investigation of collagen-GAG regenerative scaffolds in a diabetic mouse model

507 LOP31: Peripheral nerve repair: Multimodal comparison of the regenerative potential of adipose tissue derived cells in a biodegradable conduit *P. E. Engels, E. A. Kappos, M. Meyer zu Schwabedissen, M. Tremp, A. Fischmann, D. J. Schaefer, D. F. Kalbermatten University Hospital Basel, Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, Basel, Switzerland University Hospital Basel, Department of Radiology, Basel, Switzerland

Abstract 1429: Apc heterozygosity in the C57BL/6J-Min/+ mouse produces an abnormal exuberant proliferative response in excisional wound healing

The Min mouse is a model for intestinal tumor formation. Previously, we showed that the ApcMin allele produces a dominant-negative effect on enterocyte migration and stromal interaction in vivo. Tumorigenesis is driven by features of epithelial-mesenchymal interaction that are observed in wound healing, therefore we hypothesized that the Min phenotype also involved abnormalities in the wounding response program. We performed excisional wound assays on female Min mice (10 or 20-week old), comparing response to that of female age-matched Apc+/+ (WT) mice. The healing kinetics of full thickness dorsum wounds were quantified by digital planimetry. Histological cross sections on post-operative day 7 were used to measure granulation tissue thickness, nascent blood vessel density, and cell proliferation. Western blotting was performed using granulation tissue lysates to compare the expression of adhesion and extracellular matrix proteins, as well as α-smooth muscle actin, a marker for the presence of contractile cells. Immunofluorescent histochemistry was performed to detect the co-expression and quantitation of αSMA+, vimentin+, SM22+, desmin− myofibroblasts at wound edges. Wound closure was faster in Min mice relative to WT mice, 88.85% versus 68.86% (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2011-1429