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Cardiac and vascular diseases represent one of the most substantial medical areas for the applications of regenerative medicine. Despite advances in endovascular repair, surgical intervention, and disease management, atherosclerosis and heart failure continue to be prominent health problems. This report analyzes the regenerative medicine treatment opportunities in both cardiovascular and peripheral vascular repair, examining the treatment opportunities for tissue-engineered vascular grafts as well as cell-based therapies. U.S. hospital discharge data were used to generate a detailed estimate of the relative target populations for cardiac and vascular disease. Gap analyses were performed for vascular access, small caliber vascular grafts, and cell-based therapies for revascularization and heart failure. The analysis compared current alternatives, gaps in medical need, and what a tissue-engineered or regenerative alternative should achieve for optimum medical and commercial feasibility. Although the number of coronary bypass grafts vastly outnumbered peripheral grafts, a detailed consideration of re-grafts and the success of first grafts combined with gap analysis (GAP) leads us to conclude that peripheral vascular disease is the more commercially feasible and attractive target opportunity for engineered small caliber grafts for the foreseeable future. Cardiac bypass would need substantial long-term clinical experience, which could be a significant hurdle. Vascular access, often regarded as a first-in-man indication, is an excellent opportunity for an engineered graft as an alternative to arteriovenous fistula that could overcome complications associated with a prosthetic graft. The GAP also suggests that for heart failure, cellular therapies should link near-term changes in repair, such as improvement in cardiac output and reduced scarring with limiting progression of the disease, reducing the need for complex pharmacologic management, and reducing rates of hospitalization. Naturally, researchers must determine where their technology and know-how can be applied most effectively, but it is clear from our analysis that an astute strategy in the use of science and technology will be important to successful translation in this space.

The processes behind tissue response to injury and innate immunity are integral parts of the acute wound response and the initiation of repair. In addition, inflammation is a key factor influencing both positive and negative aspects of healing in chronic wounds. Biological data on the signaling mechanisms behind these basic processes has increased dramatically over the last 10 years, yet the products and practice of wound healing have not benefited to the fullest extent from this new knowledge. An in-depth analysis of the biological mechanisms underpinning the processes that impact healing was undertaken to discover ways this information might be used to improve the treatment of chronic wounds. A synopsis of findings is presented regarding the biological mechanisms at work in injury and inflammation. It examines the early stages of wound healing from a mechanistic, biological perspective to gain insight into how this information might translate to the better use and development of wound healing products. These biological processes can impact the effectiveness of treatment from wound bed preparation to potentially regenerative products like growth factors and bioengineered skin constructs. The authors conclude that approaching wound healing from the perspective of biological mechanism can improve how effectively wounds are treated today. As importantly, viewing the development of wound care and wound healing products from the perspective of biological mechanism can lead to new ways of treating wounds that achieve greater clinical significance.

Background Recently, several ligand interactions have been examined in detail as potential mediators of costimulatory signaling. The CD154/CD40 and CD28/B7 interactions have been highlighted as being among the more-significant contributors to proper activation of unprimed T lymphocytes. Human keratinocytes (HK) and human dermal fibroblasts (HF) are capable of expressing Class II HLA and CD40 antigens after interferon-gamma exposure, yet neither express significant levels of B7. HK and HF have been characterized as "nonprofessional" antigen presenting cells (APC) and their poor APC function has been partially attributed to deficient costimulatory activity. Methods In this study, we examined whether substituting for costimulatory signaling events through the addition of cross-linked monoclonal antibodies against the T-cell ligand/s (CD28 and/or CD154) could restore allostimulation. Mixed lymphocyte reactions were performed combining enriched human peripheral blood T cells and allogeneic HK or HF with or without stimulatory anti-CD28 and/or anti-CD154 antibodies. Results The results show that the addition of anti-CD28 alone permitted HF but not HK to present alloantigen effectively. In contrast, addition of both anti-CD154 and anti-CD28 was required to generate even a moderate proliferative response to allogeneic HK. Further, adding a monomorphic anti-HLA-DR antibody substantially inhibited these responses. Additional experiments suggest that signaling through CD40/CD154 directs HK to produce TGF-beta, which would adversely affect T-cell activation. Conclusions The data presented highlight significant differences in signaling capacities for HK versus HF and provide evidence for a partial mechanism by which allogeneic human skin equivalents might be immunologically null upon engraftment.

Porcine intestinal collagen (ICL), derived from processed small intestine, is used as a part of a remodelable bilaminate biosynthetic vascular prosthesis. The process for the production of ICL involves mechanical cleaning of non-crosslinked porcine intestine (NC-ICL), disinfection with peracetic acid (PA-ICL), and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (PA/EDC-ICL). Two model systems were investigated to evaluate the effect of these agents on the humoral response to NC-ICL. First, the antibody titers of rabbits immunized with NC-ICL, PA-ICL, and PA/EDC-ICL were determined, and second, the humoral response of canines receiving collagenous vascular implants was examined. Collagenous and noncollagenous fractions were extracted from NC-ICL, PA-ICL, and PA/EDC-ICL and separated by SDS-PAGE. PA and EDC treatment decreased the number of extractable proteins as compared to NC-ICL. Immunoblot techniques demonstrated anti-NC-ICL antibodies recognized multiple immunoreactive proteins in NC-ICL, but not in PA-ICL or PA/EDC-ICL; and rabbits immunized with NC-ICL produced higher antibody titers to ICL proteins than rabbits immunized with either PA-ICL or PA/EDC-ICL. It was, therefore, apparent that NC-ICL was more antigenic than either PA-ICL or PA/EDC-ICL. The humoral immune response of canines to PA/EDC-ICL fabricated vascular grafts was determined. At 4 weeks, 8 weeks, and 12 weeks postimplant, serum antibodies to ICL proteins or type I collagen could not be detected. These data demonstrate a reduced humoral immune response to PA/EDC-ICL. (c) 1996 John Wiley & Sons, Inc.

Background. Angiogenesis is characteristic of chronic inflammatory reactions. The process of angiogenesis is reported to be proinflammatory in part due to enhanced adhesion events and in part due to increased perfusion and permeability to sites of inflammation. However, little is known about the association between angiogenesis and rejection. Methods. Severe combined immune deficient mice are permissive for the growth of human skin allografts and human peripheral blood mononuclear cells (PBMC). Human PBMC were injected into mice by intravenous or intraperitoneal injection. The infiltration of cells and the associated angiogenesis reactions in the skin allografts were analyzed temporally by videomicroscopy and spatially by immunohistochemistry. Results. Human alloreactive mononuclear cells migrated to human skin but not mouse skin within hours after the intravenous infusion of PBMC. Within 3 days, areas of angiogenesis were observed in the skin grafts at the sites of infiltrates. The vessel densities in skin grafts were 24±6 vessels per calibrated grid at baseline on the day of the infusion and increased to 55±16 vessels per calibrated field by day 10. Skin grafts harvested from humanized severe combined immune deficient mice 7-14 days after the intraperitoneal infusion of human PBMC showed a similar increased density of vessels that were spatially associated with mononuclear cell infiltrates. Conclusions. A significant angiogenesis response was associated with the cell infiltrates in the human skin allografts. The onset of angiogenesis appeared after the initial development of localized infiltrates and preceded the development of microvascular destruction. These findings suggest that alloreactive T cells and/or monocytes mediate the angiogenesis response in skin allografts.

Background Engineered tissues have been proposed for the treatment of a variety of conditions including the partial or complete replacement of human organs. To determine the basis for the rejection of these tissues, we analyzed the immune response to allogeneic human skin equivalent (HSE, also called Apligraf) in the humanized SCID mouse (hu-PBL-SCID). Methods Two models of hu-PBL-SCID were used for these studies. In one model, human skin or HSE was transplanted onto humanized mice so that graft survival could be analyzed. In the other model, skin grafts were allowed to heal on naive mice before humanization. This model was used to analyze the immunologic response to the vascularized skin allograft. Humanization was performed by adoptive transfer of human PBL into SCID mice by i.p. injection. Results Both human foreskin and HSE successfully engrafted onto naive SCID mice and remained stable for more than 6 months. In contrast, human foreskin was rejected by 21 days posttransplant in hu-PBL-SCID, whereas HSE consistently engrafted for more than 28 days. Treatment of HSE grafts with interferon-y for 5 days to induce maximal MHC class II molecule expression before grafting failed to induce rejection. HSE also engrafted onto hu-PBL-SCID mice that were exposed to alloantigen by prior injection with interferon-gamma-treated keratinocytes identical to those used to generate the HSE. In addition, we determined that humanization of SCID mice following engraftment and vascularization of human foreskin resulted in marked CD3+ T cell infiltrates and a lymphocyte-induced vasculitis. In contrast, the response in vascularized HSE was associated with minimal CD3+ T cell infiltration in the absence of vasculitis or morphological features of rejection. Conclusion These results support the use of HSE and other allogeneic engineered tissues in humans provided that such tissues are limited in their antigen presenting capabilities. In addition, our findings suggest a critical function for the donor endothelial cell in rejection.

Background. Tissue-engineered products are usually composed of living cells and their supporting matrices that have been grown in vitro, using a combination of engineering and life sciences principles. Apligraf is a bilayered product composed of neonatal-derived dermal fibroblasts and keratinocytes, and Type I bovine collagen. Objective. To evaluate in a prospective, multicentered open study, the effects of tissue therapy with a tissue-engineered skin (Apligraf) with partial or full-thickness excisional wounds. Methods. One hundred and seven patients participated in this study. The tissue-engineered skin was applied once, immediately after excisional surgery, usually for skin cancer, and patients were followed for up to one year. Results. The safety results were impressive, with no clinical or laboratory evidence of rejection. Clinically, graft persistence was good to excellent in 77 of 105 (73.3%) of patients at one week, falling to 56.6% and 53.6% at two weeks and one month respectively. Conclusion. To date, this is the largest experience with a tissue-engineered skin product in acute wounds, and this study suggests that tissue therapy may be safe and useful.

Recent investigations in antigen processing suggest that many hematopoietic and nonhematopoietic cell types are capable of presenting alloantigen to T lymphocytes. However, the role of certain nonclassical antigen presenting cells is blurred by their apparent ability to down-regulate the immune response as well as activate immune cells, depending upon the microenvironment and the functional state of the responding cells. In this study we examine the ability of cultured allogeneic keratinocytes to inhibit the response of naive T cells to alloantigen or to anti-CD3. Our results demonstrate that as few as 6.25 × 10 3 keratinocytes significantly inhibited T cell proliferation in response to alloantigen as well as anti-CD3-mediated stimulation (49 and 54%, respectively). HK-mediated inhibition of T cell proliferation did not require cell contact, suggesting that inhibition is mediated by cytokines or other soluble factors. This was further supported by experiments demonstrating the inducibility of HK inhibitory activity in the presence of FCS, and the partial blockage of HK inhibitory activity through the addition of indomethacin or anti-TGFβ antibody. Interestingly, the data suggest that IL-10, a known immunomodulatory cytokine, does not play a role in the inhibitory activity seen in this system. Taken together the results suggest that HK have the potential to regulate the response of T cells to antigen presented by other APC through the production of soluble factors.

Regenerative therapies possess high theoretical potential for medical advance yet their success as commercial therapeutics is still open to debate. Appropriate data on target opportunities that provide perspective and enable strategic decision making is necessary for both efficient and effective translation. Up until now, this data have been out of reach to research scientists and many start-up companies—the very groups currently looked to for the critical advance of these therapies. The target-based estimate of opportunity presented in this report demonstrates its importance in evaluating medical need and technology feasibility. In addition, analysis of U.S. research spending, productivity, and innovation reveals that U.S. basic research in this field would benefit from greater interdisciplinarity. Overcoming the barriers that currently prevent translation into high value therapies that are quickly clinically adopted requires simultaneous integration of engineering, science, business, and clinical practice. Achieving this integration is nontrivial.

A novel injectable human dermal matrix has been developed for the treatment of complex diabetic sinus tract wounds. Bioengineered grafts are commercially available that have been somewhat effective in treating chronic wounds such as diabetic foot ulcers; however, these bioengineered grafts are only available in sheet form. These therapies are less effective in treating complex or irregularly shaped wounds that demonstrate tunnels or extensions into deep soft tissue. One acellular graft (GRAFTJACKET, Matrix, Wright Medical Technology, Arlington, Tennessee) that has been shown to effectively treat open wounds is also available in a micronized form (GRAFTJACKET Xpress Scaffold, Wright Medical Technology). This human dermal graft forms a flowable soft tissue scaffold that can be delivered via syringe into tunneling wounds. In this retrospective series, 12 patients with deep tunneling wounds were treated with GRAFTJACKET Xpress Scaffold and followed for 12 weeks. Complete wound healing was achieved in 10 of 12 patients within the 12-week evaluation. The average time to complete healing was 8.5 weeks, whereas the average time to depth healing was 7.8 weeks. The data from the study suggest that this injectable human dermal matrix has unique properties that allow it to facilitate healing of complex tunneling diabetic foot ulcers. The material is easy to prepare and inject into the wound, thereby preventing the necessity of extensive surgical exposure. The matrix supports neo-subcutaneous tissue formation and allows the body to rapidly repair these wounds.

The need for human tissue to aid in organ repair or provide a curative therapy is well known. In this review, we discuss the properties of the epidermal keratinocyte progenitor cell and the biology that underlies the methods that have helped deliver cell therapies to the clinic using this cell type. In addition, we review what the keratinocyte and the dermal fibroblast have taught us about the potential immunogenicity of allogeneic cells. The many observations made using the keratinocyte have broader biological implications and we discuss how this body of work parallels neural stem cell culture and might help us interpret cell behavior in the pancreas.

The "engineering" of a tissue implies that it can be constructed by assembling the necessary components. However, tissues are formed through an evolving, interactive process, not through a collection of parts. This chapter focuses on the biology of the progenitor cell, the native precursor to new tissue, and its role in neogenesis, or the de novo generation of functional tissue. We present a working hypothesis for the generation of parenchymal cell populations and use this hypothesis as a basis for analysis of three parenchymal populations, epidermal cells, hepatocytes of the liver, and pancreatic islets, with a view toward what impact this information will have on the development of cell therapies. By comparing developmental processes, response to injury and disease, and behavior in vitro, we conclude that the adult progenitor cell retains the potential for substantial growth and organ neogenesis and that its biological properties make it the cell of first choice for the engineering of tissues.

Objective To compare the behavior of a tissue-engineered living skin equivalent (LSE) with an autograft in acute donor site wounds. Design Paired-comparison, randomized control trial. Setting A university dermatology service. Patients Three donor sites were created on the anterior thigh of each of 20 patients requiring split-thickness skin grafts. Intervention For each patient, the donor sites were randomly assigned to be treated with meshed LSE, meshed autograft, or a polyurethane film (PUF) occlusive dressing. Blood and biopsy samples were taken for immunologic and histological studies. Main Outcome Measures Toxic effects or clinically apparent rejection, humoral and cellular immune responses, clinical take, healing time, pain, and 1-month histological appearance. Results There was no toxic effect or clinically apparent rejection of LSE. Results of humoral and cellular studies were unchanged from baseline. The average time to healing for LSE with clinical take was 7.3 days (SD,±0.8 days); for autograft, 7.6 days (SD,±1.1 days); and for PUF, 9.5 days (SD,±1.8 days). The difference between LSE or autograft and PUF was statistically significant at the .001 level. Pain was experienced by 1 patient, no patients, and 10 patients at the LSE, autograft, and PUF sites, respectively. Histologically, LSE had the thickest epidermis ( P =.02), PUF had the greatest degree of fibrosis ( P =.02), and autograft had the least degree of increased inflammation ( P =.004) and vascularity ( P =.01). Conclusions In acute donor site wounds, LSE appeared to clinically take and to be a safe and usable form of tissue therapy.