Myogel supports the ex-vivo amplification of corneal epithelial cells

Limbal stem cell deficiency leads to conjunctivalisation of the cornea and subsequent loss of vision. The recent development of transplantation of ex-vivo amplified corneal epithelium, derived from limbal stem cells, has shown promise in treating this challenging condition. The purpose of this research was to compare a variety of cell sheet carriers for their suitability in creating a confluent corneal epithelium from amplified limbal stem cells. Cadaveric donor limbal cells were cultured using an explant technique, free of 3T3 feeder cells, on a variety of cell sheet carriers, including denuded amniotic membrane, Matrigel, Myogel and stromal extract. Comparisons in rate of growth and degree of differentiation were made, using immunocytochemistry (CK3, CK19 and ABCG2). The most rapid growth was observed on Myogel and denuded amniotic membrane, these two cell carriers also provided the most reliable substrata for achieving confluence. The putative limbal stem cell marker, ABCG2, stained positively on cells grown over Myogel and Matrigel but not for those propagated on denuded amniotic membrane. In the clinical setting amniotic membrane has been demonstrated to provide a suitable carrier for limbal stem cells and the resultant epithelium has been shown to be successful in treating limbal stem cell deficiency. Myogel may provide an alternative cell carrier with a further reduction in risk as it is has the potential to be derived from an autologous muscle biopsy in the clinical setting

Macrophage phenotype in response to implanted synthetic scaffolds : An immunohistochemical study in the rat

Macrophages predominate among the cells that directly interact with biomaterials and are key orchestrators of host-biomaterial interactions. However, the macrophage response to synthetic scaffolds in particular has not been well studied. The aim of this study was therefore to characterise the macrophage response to several synthetic scaffolds in the rat using immunohistological techniques for a panel of markers of macrophage subclass or activation, including ED1 (CD68), ED2 (CD163), CD80, mannose receptor and inducible nitric oxide synthase (iNOS). Materials were implanted subcutaneously and collected after 6-8 weeks during the chronic phase of the host response. Unmodified polycaprolactone scaffolds uniquely demonstrated a total lack of both macrophage adherence to surfaces and a wider foreign body response compared to scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and polyurethanes (PURs), with those macrophages present having a clear M2 (MR+, CD80-, iNOS-) phenotype. PLGA scaffolds displayed an M1-dominant (CD80+, iNOS+, MR-) response with substantial foreign body giant cell (FBGC) formation, whilst PUR scaffold FBGCs had a more mixed M1 (CD80+, iNOS+) and M2 (MR+) phenotype. The study also identified that the use of the ED1 antibody in the rat as a pan-macrophage marker is problematic as there is a separate and substantial ED2-positive macrophage population that it does not label, both in response to biomaterials and in normal tissues. The biomaterial-dependent nature of activation for both macrophages and FBGCs was confirmed, and nuanced M1/M2 phenotypes were described

Role of NADPH oxidase in tissue growth in a tissue engineering chamber in rats

Previously we described a subcutaneous arteriovenous loop (AVL)-based tissue engineering chamber system, which contains an intrinsic circulation circuit created by joining the proximal ends of the femoral artery and vein with a venous graft. We showed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was involved in mediating neovascularization inside the chamber. However, the role of NADPH oxidase in tissue formation in the chamber is unknown. In this study, we examined the effects of gp91ds-tat, a peptidyl inhibitor of NADPH oxidase, on the growth of engineered tissue blocks, using a rat chamber model. Chambers containing the AVL were filled with Matrigel mixed with gp91ds-tat (100 µM) or the scrambled control peptide. At 14 days, in control chambers, most of the Matrigel was replaced by granulation tissues; gp91ds-tat treatment significantly reduced the level of reactive oxygen species and retarded the tissue formation process. Although the total number of blood vessels per unit cellularized area was not different between two groups, most vessels in gp91ds-tat-treated tissues had smaller lumens as compared to control. The total area occupied by vessel lumens was much less in gp91ds-tat-treated tissues (10.3 ± 1.3% in control vs. 1.7 ± 0.5% in gp91ds-tat group; p < 0.001). In vitro, gp91ds-tat treatment reduced proliferation and migration of cultured microvascular endothelial cells. Our data suggest that inhibition of NADPH oxidase function retards tissue formation in the tissue engineering chamber, which may be related to the malformed new blood vessels in the engineered tissue

Intrinsics and dynamics of fat grafts: an in vitro study

Background: Despite a revived interest in fat grafting procedures, clinicians still fail to demonstrate clearly the in vivo behavior of fat grafts as a dynamic tissue substitute. However, the basic principles in cellular biology teach us that cells can survive and develop, provided that a structural matrix exists that directs their behavior. The purpose of this in vitro study was to analyze that behavior of crude fat grafts, cultured on a three-dimensional laminin-rich matrix. Methods: Nonprocessed, human fat biopsy specimens (approximately 1 mm) were inoculated on Matrigel-coated wells to which culture medium was added. The control group consisted of fat biopsy specimens embedded in medium alone. The cellular proliferation pattern was followed over 6 weeks. Additional cultures of primary generated cellular spheroids were performed and eventually subjected to adipogenic differentiation media. Results: A progressive outgrowth of fibroblast-like cells from the core fat biopsy specimen was observed in both groups. Within the Matrigel group, an interconnecting three-dimensional network of spindle-shaped cells was established. This new cell colony reproduced spheroids that functioned again as solitary sources of cellular proliferation. Addition of differentiation media resulted in lipid droplet deposition in the majority of generated cells, indicating the initial steps of adipogenic differentiation. Conclusions: The authors noticed that crude, nonprocessed fat biopsy specimens do have considerable potential for future tissue engineering based applications, provided that the basic principles of developmental, cellular biology are respected. Spontaneous in vitro expansion of the stromal cells present in fat grafts within autologous and injectable matrices could create "off-the-shelf" therapies for reconstructive procedures. (Plast. Reconstr. Surg. 126: 1155, 2010.

An adipogenic gel for surgical reconstruction of the subcutaneous fat layer in a rat model

‘Off-the-shelf’ tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRβ-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice

An adipoinductive role of inflammation in adipose tissue engineering: Key factors in the early development of engineered soft tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue