Bi-layered living cellular construct resulted in greater healing in an alloxan-induced diabetic porcine model.

Tissue-engineered skin constructs, including bi-layered living cellular constructs (BLCC) used in the treatment of chronic wounds, are structurally/functionally complex. While some work has been performed to understand their mechanisms, the totality of how BLCC may function in wound healing remains unknown. To this end, we have developed a delayed wound healing model to test BLCC cellular and molecular mechanisms of action. Diabetes was chemically-induced using alloxan in Yucatan miniature pigs, and full-thickness wounds were generated on their dorsum. These wounds were either allowed to heal by secondary intention alone (control) or treated with a single or multiple treatments of a porcine autologous BLCC. Results indicated a single treatment with porcine BLCC resulted in statistically significant wound healing at day 17, while four treatments resulted in statistically significant healing on days 10, 13, and 17 compared to control. Statistically accelerated wound closure was driven by re-epithelialisation rather than contraction or granulation. This porcine diabetic model and the use of a porcine BLCC allowed evaluation of healing responses in vivo without the complications typically seen with either xenogenic responses of human/animal systems or the use of immune compromised animals, expanding the knowledge base around how BLCC may impact chronic wounds.

A graft model for hair development.

Follicular cell implantation (FCI) is an experimental cell therapy for the treatment of hair loss that uses cultured hair follicle cells to induce new hair formation. This treatment is based on the demonstration that adult dermal papilla cells (DPC) retain the hair inductive capacity they acquired during hair morphogenesis in the embryo. For FCI, hair inductive cells are isolated from scalp biopsies and then propagated in culture in order to provide enough cells to generate many new follicles from a few donor follicles. Following expansion in culture, the cells are implanted into the scalp where they induce the formation of new follicles. Because the process relies on the ability to retain the potential for hair induction during the expansion of DPC in culture, we sought a consistent, reliable and easily performed in vivo assay in which to test hair induction. In this study, we describe a simple graft model that supports hair morphogenesis. The assay combines dermal cells with embryonic mouse epidermis that provides the keratinocyte component of induced follicles. The grafts are placed under a protective skin flap in the host athymic mouse where the cells will form a skin graft with hair if the dermal cells are hair inductive DPC. Using the assay, freshly isolated and cultured mouse embryo dermal cells as well as cultured dermal papilla cells from other species all induced hair formation. The induced hairs were aesthetically indistinguishable from those of the epidermal donor in length, thickness, and pigmentation, and they were histologically normal.

Bioengineered Self-assembled Skin as an Alternative to Skin Grafts.

For patients with extensive burns or donor site scarring, the limited availability of autologous and the inevitable rejection of allogeneic skin drive the need for new alternatives. Existing engineered biologic and synthetic skin analogs serve as temporary coverage until sufficient autologous skin is available. Here we report successful engraftment of a self-assembled bilayered skin construct derived from autologous skin punch biopsies in a porcine model. Dermal fibroblasts were stimulated to produce an extracellular matrix and were then seeded with epidermal progenitor cells to generate an epidermis. Autologous constructs were grafted onto partial- and full-thickness wounds. By gross examination and histology, skin construct vascularization and healing were comparable to autologous skin grafts and were superior to an autologous bilayered living cellular construct fabricated with fibroblasts cast in bovine collagen. This is the first demonstration of spontaneous vascularization and permanent engraftment of a self-assembled bilayered bioengineered skin that could supplement existing methods of reconstruction.

Survival of Allogeneic Self-Assembled Cultured Skin.

BACKGROUND: Deficiency of autologous skin for reconstruction of severe wounds is a major problem in plastic surgery. Autologous substitutes can provide additional coverage, but due to the duration of production, treatment is significantly delayed. The allogeneic approach offers a potential of having an off-the-shelf solution for the immediate application. METHODS: In this study, we assess the engraftment and immunogenicity of allogeneic bilayered bioengineered skin prepared by a self-assembly method. Bioengineered skin has the potential immunological advantage of lacking passenger leukocytes including antigen-presenting cells. The skin constructs were transplanted across major histocompatibility complex (MHC) barriers in a porcine animal model. Animals received a second grafting of the same skin construct 7 weeks after the first set of grafts together with MHC-matched constructs to assess for clinical sensitization. RESULTS: All alloconstructs successfully engrafted with histologic evidence of neovascularization by day 4. Complete cellular rejection and tissue loss occurred by day 8 for most grafts. After the second application, accelerated rejection (<4 days) took place with the development of swine MHC-specific cytotoxic alloantibody. CONCLUSIONS: These data demonstrate preclinically that self-assembled allogeneic constructs engraft and reject similar to allogeneic skin despite the absence of professional donor antigen-presenting cells.

Hair follicle neogenesis induced by cultured human scalp dermal papilla cells.

AIM: To develop a method by which human hair follicle dermal papilla (DP) cells can be expanded in vitro while preserving their hair-inductive potential for use in follicular cell implantation, a cellular therapy for the treatment of hair loss. MATERIALS & METHODS: DP cells were isolated from scalp hair follicles in biopsies from human donors. DP cell cultures were established under conditions that preserved their hair-inductive potential and allowed for significant expansion. The hair-inductive potential of cells cultured for approximately 36 doublings was tested in an in vivo flap-graft model. In some experiments, DiI was used to label cells prior to grafting. RESULTS: Under the culture conditions developed, cultures established from numerous donors reproducibly resulted in an expansion that averaged approximately five population doublings per passage. Furthermore, the cells consistently induced hair formation in an in vivo graft assay. Grafted DP cells appeared in DP structures of newly formed hairs, as well as in the dermal sheath and in the dermis surrounding follicles. Induced hair follicles persisted and regrew after being plucked 11 months after grafting. CONCLUSION: A process for the propagation of human DP cells has been developed that provides significant expansion of cells and maintenance of their hair-inductive capability, overcoming a major technical obstacle in the development of follicular cell implantation as a treatment for hair loss.

Hair morphogenesis in vitro: formation of hair structures suitable for implantation.

AIM: To develop a construct through which implanted follicular cells will efficiently cause hair regeneration for the treatment of androgenetic alopecia. MATERIALS & METHODS: Follicular dermal and epidermal cells isolated from embryonic mouse skin were formed into aggregates. The aggregates were incubated in culture for 5-7 days and then implanted intradermally into athymic mice. RESULTS: During culture, mixed cell aggregates developed into hair-like structures, termed 'proto-hairs'. Proto-hairs contained structures that resembled normal hair components, such as dermal papillae, hair matrix and rudimentary hair shafts. When implanted into mouse skin, they developed further into mature hair follicles capable of prolonged growth. CONCLUSION: Mixed aggregates of murine follicular cells have the ability to develop in culture into proto-hairs that retain the ability to fully develop into hair follicles after implantation. Proto-hairs from human cells could provide a convenient and practical means by which follicular cells could be implanted for efficient hair regeneration to treat hair loss.

Development and manufacture of an investigational human living dermal equivalent (ICX-SKN).

AIM: To design and manufacture an investigational living skin graft replacement (ICX-SKN) that is able to incorporate into the host, providing healing by primary intent without the need for a second intervention. MATERIALS & METHODS: The ICX-SKN skin graft replacement has been designed as an allogeneic dermal substitute comprising an extracellular matrix composed largely of human collagen and human dermal fibroblast cells (HDFs). ICX-SKN is first formed by casting a provisional matrix of fibrin, into which HDFs are seeded. Through a process of maturation, HDFs are induced to lay down collagen and other extracellular matrix materials and, as the construct matures, the original fibrin is largely replaced by collagen, which provides tensile strength and flexibility to the construct. In order to design a product and manufacturing system that lends itself to large-scale production the process was developed as a discontinuous process consisting of four stages: 1. batch casting and maturation of the initial construct (pSKN), 2. freeze-drying of pSKN to produce a second intermediate (dSKN), 3. sterilization by gamma-irradiation of dSKN to produce a third intermediate (sSKN), and finally, 4. repopulation of sSKN by fresh HDFs to produce the final product, ICX-SKN skin graft replacement. Preliminary characterization of ICX-SKN and its application in a preclinical model are described. RESULTS: The 7-week maturation period resulted in a construct (pSKN) with robust handling properties, which was composed mainly of human collagen I. Following development of a process for freeze-drying and subsequent sterilization, the matrix was successfully repopulated with fresh HDFs. In addition, it was demonstrated that human keratinocytes attached and differentiated on the matrix. Application of human keratinocytes to the repopulated constructs (ICX-SKN) resulted in expression of markers of basement membranes that was largely dependent on the presence of living HDFs on the constructs. ICX-SKN graft replacements applied to excision wounds in mice healed and were rapidly re-epithelialized. CONCLUSIONS: ICX-SKN has been developed as a platform product that can be used as a skin graft replacement and the process by which it is manufactured has been designed for the product to be available to the end-user off-the-shelf and for ease-of-use in practice.

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud.

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.