Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition.

The aim of this study was to determine the influence of two key scaffold design parameters, void fraction (VF) and pore size, on the attachment, growth, and extracellular matrix deposition by several cell types. Disc-shaped, porous, poly(-lactic acid) (L-PLA) scaffolds were manufactured by the TheriForm solid free-form fabrication process to generate scaffolds with two VF (75% and 90%) and four pore size distributions (< 38, 38-63, 63-106, and 106-150 microm). Microcomputed tomography analysis revealed that the average pore size was generally larger than the NaCl used, while VF was at or near the designated percentage. The response of three cell types-canine dermal fibroblasts (DmFb), vascular smooth muscle cells (VSMC), or microvascular epithelial cells (MVEC)-to variations in architecture during a 4-week culture period were assessed using histology, metabolic activity, and extracellular matrix deposition as comparative metrics. DmFb, VSMC, and MVEC showed uniform seeding on scaffolds with 90% VF for each pore size, in contrast to the corresponding 75% VF scaffolds. DmFb showed the least selectivity for pore sizes. VSMC displayed equivalent cell proliferation and matrix deposition for the three largest pore sizes. MVEC formed disconnected webs of tissue with sparse extracellular matrix at 90% VF and >38 to 150 microm; however, when cultured on scaffolds with pores formed with salt particles of <38 microm, MVEC formed a multilayered lining on the scaffolds surface. Culture data from scaffolds with a 75% VF suggests that the structural features were unsuitable for tissue formation. Hence, there were limits of acceptable scaffold architecture (VF, pore size) that modulated in vitro cellular responses.

Development and characterization of tissue-engineered aortic valves.

Tissue-engineered aortic valves, known as recellularized heart valves, were developed by seeding human neonatal fibroblasts onto decellularized, porcine aortic valves. Recellularized heart valves were cultured up to 8 weeks in a novel bioreactor that imposed dynamic pulsatile fluid flow to expose the dermal fibroblasts to mechanical forces. Our data showed that, under static or dynamic flow conditions, dermal fibroblasts attached to and migrated into the decellularized, porcine valve scaffolding. The human cells remained viable as indicated by MTT viability staining. Gradual colonization of the decellularized porcine scaffolding by the human dermal fibroblasts was shown histologically by hematoxylin & eosin staining, immunocytochemically using a monoclonal antibody directed against prolyl-4-hydroxylase (an intracellular enzyme expressed by human fibroblasts synthesizing collagen), and quantitative digital image analyses. Thymidine and proline radiolabeled analog studies at 1, 2 and 4 weeks of individual leaflets cultured statically demonstrated that the human fibroblasts were mitotic and synthesized human extracellular matrix proteins, thereby supplementing the existing porcine matrix. The overall approach results in a heart valve populated with viable human cells. In the development of valves that perform in a similar manner as natural biological structures, this approach may present some unique benefits over current medical therapies.

A model system for long-term serum-free suspension organ culture of human fetal tissues: experiments on digits and skin from multiple body regions.

We have optimized a suspension organ culture (SOC) system previously used to study human fetal skin in vitro. Eleven types of media were used to culture fetal digits and skin samples (ranging from 59-91 days of estimated gestational age) for periods of 3 or 4 weeks. The cultures were supplemented with 0%-10% bovine calf serum and exposed to 5%-45% oxygen atmospheres. Optimal conditions, which were selected based upon the morphologic and histologic properties of the cultured tissues, were: DMEM-F12 medium in a serum-free environment with a 45% oxygen atmosphere. The refined SOC system supports the maintenance and/or continued development of the epidermis and dermis of trunk, back, scalp, and digital skin, developing hair follicles, nails, and glands. Overall, development and differentiation mimicked the in vivo state more closely than that observed when using other in vitro culture systems. The effects of bovine calf serum and oxygen levels are presented, and the potential for manipulating the environment of this system for studying mechanisms of cutaneous development is also discussed.

Avian scale development. XVII: The epidermis of the scaleless (sc/sc) anterior metatarsal skin is determined, but the dermis lacks permissive cues for the patterned expression of the determined state.

Embryos homozygous for the gene scaleless (sc/sc) completely lack scutate scales and the beta strata which characterize terminal differentiation of the scale ridges located on the anterior metatarsal region of the foot. Although the sc/sc epidermis cannot undergo scale morphogenesis, it can respond to the inductive dermal ridges of normal scutate scales by generating beta strata. Recently, we discovered that the anterior metatarsal epidermis of normal embryos becomes committed to the formation of beta strata prior to morphogenesis of definitive scale ridges. Here, we examined the possibility that the sc/sc anterior metatarsal epidermis also becomes determined, i.e., committed to scutate scale-specific terminal differentiation. Experimental tissue recombinants were used to assess the ability of the sc/sc epidermis to generate beta strata. The results show that the germinative cells of the 15-day sc/sc epidermis are committed to generating beta strata, even though they have not undergone scutate scale morphogenesis. Thus, the mechanisms involved in establishing epidermal determination must differ form those regulating scale morphogenesis. In addition, we examined the formation of patterned, permissive cues in the anterior metatarsal and footpad dermises of sc/sc embryos. Analysis of recombinants showed that both the 15- and 20-day dermises from the sc/sc anterior metatarsal region fail to provide cues for beta stratum formation, when associated with the determined 15-day scutate scale epidermis. Likewise, the 15-day sc/sc footpad dermis cannot support beta stratum formation. However, 20-day sc/sc footpad dermis is able to support the generation of a few abnormally patterned beta strata, demonstrating that sc/sc dermis which has experienced even limited morphogenesis is able to provide permissive cues for the terminal differentiation of the scutate scale epidermis.

Avian scale development. XVI. Epidermal commitment to terminal differentiation is prior to definitive scale ridge formation.

Germinative cells of the scutate scale epidermis from 15-day embryos are committed to appendage-specific, beta stratum formation in association with a foreign dermis. Commitment precedes the time (17 days of development) at which beta strata are actually present in their site-specific locations along the outer surface of each scutate scale. This observation suggested the possibility that commitment to beta stratum formation might be occurring as the outer epidermal surface of each scutate scale first becomes established between 12 and 13 days of development. It is at this time that the scale epidermis loses its ability to participate in feather morphogenesis and cell proliferation becomes restricted to a true stratum basale. To examined the ability of the presumptive scutate scale epidermis to generate beta strata in the absence of the inductive scutate scale dermis, scutate scale epidermis from 11-, 12-, and 13-day embryos was recombined with 15-day reticulate scale dermis and grown for 7 or 9 days. The dermis of reticulate scales does not induce beta stratum formation, but it does support differentiation of a beta stratum by the determined 15-day scutate scale epidermis. Using immunohistological and biochemical analyses of beta-keratins, we find that each of these presumptive scutate scale epidermises is competent to generate appendage-specific beta strata in the absence of the scutate scale dermis. This determination is occurring prior to scale ridge morphogenesis and differentiation of the epidermis into the distinct outer and inner epidermal surfaces of the scale ridge. The restricted distribution of beta strata to the apical domes of individual reticulate-like scales demonstrates that the germinative cells of the committed epidermises are responding to patterned cues. This study also suggests that all basal cells of the presumptive scutate scale epidermis are initially endowed with the ability to generate cells that form a beta stratum.

Avian scale development. XIII. Epidermal germinative cells are committed to appendage-specific differentiation and respond to patterned cues in the dermis.

The ability of the germinative cell population of scutate scale epidermis to continue to generate cells that undergo their appendage-specific differentiation (beta stratum formation), when associated with foreign dermis, was examined. Tissue recombination experiments were carried out which placed anterior metatarsal epidermis (scutate scale forming region) from normal 15-day chick embryos with either the anterior metatarsal dermis from 15-day scaleless (sc/sc) embryos or the dermis from the metatarsal footpad (reticulate scale forming region) of 15-day normal embryos. Neither of these dermal tissues are able to induce beta stratum formation in the simple ectodermal epithelium of the chorion, however, the footpad dermis develops an appendage-specific pattern during morphogenesis of the reticulate scales, while the sc/sc dermis does not. Morphological and immunohistological criteria were used to assess appendage-specific epidermal differentiation in these recombinants. The results show that the germinative cell population of the 15-day scutate scale epidermis is committed to generating suprabasal cells that follow their appendage-specific pathways of histogenesis and terminal differentiation. Of significance is the observation that the expression of this determined state occurred only when the epidermis differentiated in association with the footpad dermis, not when it was associated with the sc/sc dermis. The consistent positioning of the newly generated beta strata to the apical regions of individual reticulate-like appendages demonstrates that the dermal cues necessary for terminal epidermal differentiation are present in a reticulate scale pattern. The observation that beta stratum formation is completely missing in the determined scutate scale epidermis when associated with the sc/sc dermis adds to our understanding of the sc/sc defect. The present data support the conclusion of earlier studies that the anterior metatarsal dermis from 15-day sc/sc embryos lacks the ability to induce beta stratum formation in a foreign epithelium. In addition, these observations evoke the hypothesis that the sc/sc dermis either lacks the cues (generated during scutate and reticulate scale morphogenesis) necessary for terminal differentiation of the determined scutate scale epidermis or inhibits the generation of a beta stratum.