Collagen-glycosaminoglycan Matrix Research Articles

Collagen-GAG substrate enhances the quality of nerve regeneration through collagen tubes up to level of autograft.

Peripheral nerve regeneration was studied across a tubulated 10-mm gap in the rat sciatic nerve using histomorphometry and electrophysiological measurements of A-fiber, B-fiber, and C-fiber peaks of the evoked action potentials. Tubes fabricated from large-pore collagen (max. pore diameter, 22 nm), small-pore collagen (max. pore diameter, 4 nm), and silicone were implanted either saline-filled or filled with a highly porous, collagen-glycosaminoglycan (CG) matrix. The CG matrix was deliberately synthesized, based on a previous optimization study, to degrade with a half-life of about 6 weeks and to have a very high specific surface through a combination of high pore volume fraction (0.95) and relatively small average pore diameter (35 microm). Nerves regenerated through tubes fabricated from large-pore collagen and filled with the CG matrix had significantly more large-diameter axons, more total axons, and significantly higher A-fiber conduction velocities than any other tubulated group; and, although lower than normal, their histomorphometric and electrophysiological properties were statistically indistinguishable from those of the autograft control. Although the total number of myelinated axons in nerves regenerated by tubulation had reached a plateau by 30 weeks, the number of axons with diameter larger than 6 microm, which have been uniquely associated with the A-fiber peak of the action potential, continued to increase at substantial rates through the completion of the study (60 weeks). The kinetic data strongly suggest that a nerve trunk maturation process, not previously reported in studies of the tubulated 10-mm gap in the rat sciatic nerve, and consisting in increase of axonal tissue area with decrease in total tissue area, continues beyond 60 weeks after injury, resulting in a nerve trunk which increasingly approaches the structure of the normal control.

Vascularized collagen-glycosaminoglycan matrix provides a dermal substrate and improves take of cultured epithelial autografts.

Cultured epithelial autografts are an important adjunct in treating severely burned patients, greatly expanding the epidermis using a small donor site. Problems with cultured epithelial autografts include the time delay to culture cells to confluence and variable take on full-thickness wounds. Dermal allografts have been used as a substrate to improve the take of cultured epithelial autografts. This study examined the effect of a vascularized collagen-glycosaminoglycan matrix as a substrate for cultured epithelial autografts. The matrix was grafted onto 12 full-thickness wounds in Yorkshire pigs and allowed to vascularize for 10 days. The cultured epithelial autografts were applied over the vascularized collagen-glycosaminoglycan matrix (n = 12) or onto freshly excised full-thickness wounds (n = 10). Gross and histologic observations were made over a 3-week period. Gross observations at 7 days indicated cultured epithelial autografts to have nearly complete confluence when applied to wounds treated by collagen-glycosaminoglycan, whereas cultured epithelial autografts applied to freshly excised wounds did not take. Gross determination of epithelial confluence was verified by histologic analysis of randomly selected wounds. Histologic epithelial confluence of cultured epithelial autografts on collagen-glycosaminoglycan (98 +/- 4 percent) was significantly greater than that on full-thickness wounds (4 +/- 10 percent). Electron microscopy of the cultured epithelial autografts/collagen-glycosaminoglycan construct demonstrated anchoring fibrils at the dermal-epidermal junction at day 7. The neoepidermis of wounds treated by cultured epithelial autografts/collagen-glycosaminoglycan was hyperplastic at day 7 but developed a normal maturation sequence by 21 days. Results from this study suggest that vascularized collagen-glycosaminoglycan matrices produce a favorable substrate for cultured epithelial autografts and may improve cultured epithelial autografts take in burn patients.

Organized Skin Structure Is Regenerated In Vivo from Collagen-GAG Matrices Seeded with Autologous Keratinocytes

A well-characterized collagen-glycosaminoglycan matrix (CGM) that has been shown to function as a dermal analog was seeded with freshly disaggregated autologous keratinocytes and applied to full-thickness wounds in a porcine model. CGM were impregnated with 50,000 keratinocytes per cm2, a seeding density that produces a confluent epidermis within 19 d post-grafting and affords a 60-fold surface expansion of the donor epidermis. In this study, the temporal sequence of events in epidermal and neodermal formation was analyzed histopathologically and immunohistochemically from 4 to 35 d post-grafting. The epidermis was observed to form from clonal growth of individual keratinocytes into epithelial cords and islands that gradually enlarged, coalesced, differentiated to form large horn cysts, and finally reorganized at the graft surface to form a fully differentiated, normally oriented epidermis with rete ridges. Simultaneously, a neodermis formed from migration of endothelial cells, fibroblasts, and macrophages into the CGM from the underlying wound bed, resulting in formation of blood vessels, the production of abundant extracellular matrix, and the degradation of the CGM fibers, respectively. Gradually, the stromal cellularity of the CGM decreased and collagen deposition and remodeling increased to form a neodermal connective tissue matrix beneath the newly formed epidermis. Complete dissolution of the CGM occurred, partly as a result of degradation by an ongoing foreign-body giant cell reaction that peaked at 8-12 d post-grafting, but neither acute inflammation nor evidence of immune stimulation were observed. Within 1 mo, many structural components of normal skin were reconstituted.

Effect of keratinocyte seeding of collagen-glycosaminoglycan membranes on the regeneration of skin in a porcine model.

A collagen-glycosaminoglycan matrix, impregnated with autologous keratinocytes, was applied as island grafts onto full-thickness porcine wounds to determine whether complete epidermal coverage could be achieved in a single grafting procedure. Twenty-four grafts with seeding densities ranging from 0 to 3,000,000 cells/cm2 were used to determine the kinetics of epidermal coverage. The time sequence of epidermal formation was then studied between days 14 and 28 using four additional grafts, each seeded with a density of 500,000 cells/cm2. Autologous keratinocytes proliferated as the collagen-glycosaminoglycan matrix was vascularized to form a confluent epidermis by 2 weeks in matrices seeded with at least 100,000 cells/cm2. The epidermal thickness and the number of keratinocyte cysts observed in the neodermis at 2 weeks increased linearly with the logarithm of the seeding density. Sequential analysis of neoepidermis showed the nascent epidermis to be hyperplastic, parakeratotic, and focally lacking in granular layer differentiation at 2 weeks. After 2 weeks, it underwent normal maturation and differentiation. Irrespective of seeding density at 2 weeks the collagen-glycosaminoglycan matrix was well vascularized, contained a dense cellular infiltrate, and was almost completely degraded. These studies demonstrate that seeded keratinocytes proliferate and differentiate to form a confluent epidermis by 2 weeks in matrices seeded with at least 100,000 cells/cm2.

Healing of Tendon Defects Implanted with a Porous Collagen-GAG Matrix: Histological Evaluation

There is currently no method to restore normal function in tendon injuries that result in a gap. The objective of this study was to evaluate the early healing of tendon defects implanted with a porous collagen–glycosaminoglycan (CG) matrix, previously shown to facilitate the regeneration of dermis and peripheral nerve. A novel animal model that isolates the tendon defect site from surrounding tissue during healing was employed. This model used a silicone tube to entubulate the surgically produced tendon gap of 10 mm, allowing for the evaluation of the effects of the analog of extracellular matrix on healing of tendon, absent the influences of the external environment. The results showed that tendon stumps induced synthesis of a tissue cable inside the silicone tube in both the presence and absence of CG matrix. The presence of the CG matrix, however, altered the process of tendon healing. Tubes filled with CG matrix contained a significantly greater volume of tissue at the time periods of evaluation: 3, 6, and 12 weeks. Granulation tissue persisted for a longer period of time in the lesion site of CG-filled defects, and the amount of dense fibrous tissue increased continuously during the period of study in defects filled with CG matrix. In contrast, the amount of dense fibrous tissue decreased after 6 weeks in originally empty tubes. In tubes that did not contain the CG matrix, the new tissue consisted of dense aggregates of crimped fibers with a wavelength and fiber bundle thickness that were significantly shorter than those in normal tendon, and consistent with the type of scar that is the end result of repair of many connective tissues. Although, CG-filled tubes contained dense fibrous tissue by 12 weeks, the tissue had no crimp. The CG matrix may have prolonged the synthesis of granulation tissue and delayed or prevented the formation of scar.

Pharmacotoxicological applications of an equivalent dermis: three measurements of cytotoxicity.

The legal procedure for evaluating the toxicity of cosmetic, household, chemical and pharmaceutical products is still the irritancy Draize test on rabbits. Various irritation tests are currently being developed as alternatives to in vivo animal testing. Our in vitro model system is composed of 24 equivalent dermis (ED) comprising a chitosan-cross-linked collagen-glycosaminoglycan matrix populated by foreskin fibroblasts. In evaluating this system for irritancy testing, three different measures of toxicity were used: MTT (dimethylthiazol diphenyltetrazolium bromide) reduction, and lactate dehydrogenase and interleukin-6 release. The experiments described herein represent a preliminary evaluation to determine the usefulness and predictive value of our 24 ED kit as an alternative method for the prediction of human dermal reaction, versus three chemical products: cadmium chloride, lauryl sulfate, and benzalkonium chloride. Preliminary results suggest that the ED may be a useful in vitro model for the prediction of cutaneous and ocular toxicity and allow the development of a 24-skin-equivalent kit realized by seeding human normal keratinocytes onto the equivalent dermis.

Specific effects of glycosaminoglycans in an analog of extracellular matrix that delays wound contraction and induces regeneration

Previous studies have shown that a simple analog of extracellular matrix delays wound contraction and, if seeded with keratinocytes, inhibits scar synthesis by inducing partial regeneration of the dermis and the epidermis in full-thickness skin wounds in the guinea pig. The active extracellular matrix analog was selected from a large number of copolymers of type I collagen and chondroitin-6-sulfate differing in average pore diameter and degradation rate. However, these previous studies did not provide information on the potential role of the glycosaminoglycan component of this collagen-glycosaminoglycan matrix. The present study focuses on the effect of substitution of chondroitin-6-sulfate by other glycosaminoglycans or with the corresponding proteoglycans. The three substituents of chondroitin-6-sulfate studied were dermatan sulfate, decorin (a proteoglycan-containing dermatan sulfate chain), and aggrecan (a proteoglycan in which 90% of the glycosaminoglycan component was chondroitin-6-sulfate). Each test matrix was grafted on full-thickness skin wounds in the guinea pig, and the wound contraction kinetics were followed. Previous studies have strongly suggested that delay in onset of contraction is necessary for regeneration. Substitution of chondroitin-6-sulfate by either dermatan sulfate or decorin increased the delay in wound contraction by the greatest increment. However, the difference between substitution either by the dermatan sulfate chains or by the corresponding proteoglycan was not significant. Substitution of chondroitin-6-sulfate by aggrecan did not affect the activity of the extracellular matrix analog. These results suggest that the glycosaminoglycan component of the proteoglycan, rather than the protein core, is responsible for the increment of activity. It is speculated that the morphogenetic activity of the extracellular matrix analog resides in its putative ability to neutralize transforming growth factor-beta, leading thereby to downregulation of the inflammatory response in the wound bed.

Reconstitution of the Histologic Characteristics of a Giant Congenital Nevomelanocytic Nevus Employing the Athymic Mouse and a Cultured Skin Substitute

This study addresses the development of an animal model for human giant congenital nevomelanocytic nevi (GCNN). Skin grafts were made from 1) non-involved split-thickness skin from a 12-month-old GCNN patient, 2) nevus split-thickness skin from the same GCNN patient, 3) nevus full-thickness skin, and 4) cadaveric human split-thickness skin. For groups 1) and 2), human epidermal and dermal cells were enzymatically isolated and expanded in tissue culture. Composite grafts were made by placing the cultured dermal cells into a collagen-glycosaminoglycan (GAG) matrix, followed by placement of the epidermal cells onto the opposite, laminated side of the matrix. All grafts were placed onto full-thickness wounds of athymic mice and biopsies were obtained from 6 to 38 weeks later for light microscopy including S-100 immunoperoxidase staining, and electron microscopy. The GCNN cultured skin mice (group 2) developed black, raised skin in the healed wounds. None of the group 1 mice developed lesions, grossly or histologically. All of the nevus full-thickness mice retained the nevus grossly. Histopathologic examination at 38 weeks of the black, raised plaques of group 2 demonstrated a reconstituted dermis similar to group 3. Nevus cells were larger and more epithelioid in the upper dermis, as seen with true GCNN. These nevomelanocytes were not seen in the dermis at 24 weeks, suggesting that the nevus cells migrated from the epidermal component of the cultured graft to the dermis during this time frame (24-38 weeks). The melanocyte identity of these cells was confirmed with S-100 immunoperoxidase staining and electron microscopy. These findings are unique to this composite cultured graft system. The ability to culture specific types of melanocytes and place them int skin substitutes on athymic mice provides a basis for the study of GCNN and melanocyte biology in vivo.

Characterization of Skin Reconstructed on a Chitosan-Cross-Linked Collagen-Glycosaminoglycan Matrix

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. We have developed a reconstructed skin composed of two compartments: (1) a dermal equivalent comprising an acellular dermal substrate populated by foreskin fibroblasts and (2) an epidermis regenerated from normal human keratinocytes seeded onto the dermal equivalent. The dermal substrate contains type I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosan. Fibroblasts seeded into the porous structure of the dermal substrate provide a dermal equivalent suitable to support epidermal cells. Keratinocytes attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks of culture, histological sections show a basal layer with cuboidal cells attached to the dermal equivalent and several suprabasal cell layers including the stratum corneum. Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermoepidermal junction; however, the lamina densa was found discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. The epidermal differentiation was complete terminal with the stratum corneum containing several layers of corneocytes filled with tonofilaments. Reconstructed skin, based on our chitosan-cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for in vitro toxicological studies as well as a suitable wound covering for the treatment of patients with severe burns.

Conduction Properties in Peripheral Nerve Fibers Regenerated by Biodegradable Polymer Matrix

AbstractThe rat sciatic nerve was transected mid-thigh and grafted with a silicone tube, the central 10 mm of which was filled with a collagen-glycosaminoglycan (CG) matrix. The rats were grafted contralaterally with empty silicone tubes as controls. The earliest compound muscle action potentials (CMAP's) at the plantar muscles were recorded around 11 weeks. After 30 weeks, the distal motor latencies recovered to about 50% higher than normal, the conduction velocity to about 50% normal, and the amplitudes of the CMAP's to about 50% normal. Of 7 rats in this study, all 7 nerves grafted with the CG matrix exhibited recovery, while only 1 grafted with the empty tube exhibited recovery. The CG matrix therefore appears to promote functional nerve regeneration across extended distances.