Collagen Cell Carrier Research Articles

453 - Evaluation of a collagen cell carrier for urethral reconstructive surgery using a biological simulation model

453 - Evaluation of a collagen cell carrier for urethral reconstructive surgery using a biological simulation model

Abstract 151: Organized 3D Neo-Angiogenic and Neo-Lymphangiogenic Vascular Networks for Cardiac Regeneration

Introduction: Cardiovascular tissue engineering has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Engineering a tissue of clinically relevant magnitude requires the formation of extensive and stable microvascular networks within the tissue. Hypothesis: Functional vascularized cardiac graft can be generated by the interaction of multipotent human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-eCMs) in a 3D collagen cell carrier (CCC) scaffold. Methods and Results: To achieve the above aim, we have developed an in vitro 3D functional vascularized cardiac muscle construct using hiPSC-eCMs and hMSCs. Initially, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on CCCs for 7 days under vasculogenic culture conditions. hCMVECs/hMSCs underwent maturation and differentiation characteristic of microvessel morphogenesis and formed extensive plexuses of capillary networks. Next, the hiPSC-eCMs and hMSCs were co-cultured onto these generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. hiPSC-eCMS/hMSCs underwent maturation and differentiation, and demonstrated spontaneous rhythmic beating. The vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular and functional levels. Expression analyses of the differentiated cells revealed neo-angiogenesis and/or neo-lymphangiogenesis as well as neo-cardiomyogenesis. Conclusions: Our unique 3D co-culture system provides us the apt in vitro functional prevascularized 3D cardiac graft that can be utilized for myocardial repair and/or regeneration.

Functional Tissue Engineering: A Prevascularized Cardiac Muscle Construct for Validating Human Mesenchymal Stem Cells Engraftment Potential In Vitro.

The influence of somatic stem cells in the stimulation of mammalian cardiac muscle regeneration is still in its early stages, and so far, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific differentiated progenies, and also exhibit functional synchronization. Consequently, this necessitates the development of an appropriate in vitro three-dimensional (3D) model of cardiomyogenesis and prompts the development of a 3D cardiac muscle construct for tissue engineering purposes, especially using the somatic stem cell, human mesenchymal stem cells (hMSCs). To this end, we have created an in vitro 3D functional prevascularized cardiac muscle construct using embryonic cardiac myocytes (eCMs) and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were cocultured onto a 3D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions; hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were cocultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were characterized at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, for instance, not only were hMSCs able to couple electromechanically with developing eCMs but were also able to contribute to the developing vasculature as mural cells, respectively. Hence, our unique 3D coculture system provides us a reproducible and quintessential in vitro 3D model of cardiomyogenesis and a functioning prevascularized 3D cardiac graft that can be utilized for personalized medicine.

A Quantitative Analysis of Microcirculation in Skin Defects Covered with Topical Wound Dressings or a Newly Developed Collagen Matrix.

Innovative skin substitutes and temporary wound dressings are frequently used in the treatment of superficial and partial-thickness wounds. The aim of this study was to compare 2 commonly used temporary skin dressings with a newly developed collagen matrix to determine changes in microcirculation as measured by blood flow, hemoglobin oxygenation, and relative hemoglobin values during healing of partial-thickness skin defects. This animal study involved 28 adult male Lewis rats. On the back of each rat, 2 standardized partial-thickness skin defects were generated through a skin dermatome (n = 56). Then, wounds were treated with polylactide-based copolymer skin substitute (Suprathel, PolyMedics, Denkendorf, Germany; n = 14), biosynthetic skin dressing (Biobrane, Smith & Nephew, Hamburg, Germany; n = 14), or Collagen Cell Carrier (CCC; Naturin Viscofan, Weinheim, Germany; n = 14). The remaining control wounds were left untreated (n = 14). Perfusion dynamics were assessed every 10 days for 80 days with the O2C device (LEA Medizintechnik, Gieβen, Germany) to determine blood flow, hemoglobin oxygenation, and relative amount of hemoglobin. Blood flow was increased in all wounds for at least 30 days after wound generation. The relative amounts of hemoglobin were increased in superficial layers (2 mm) for 10 to 20 days. Hemoglobin oxygenation in the superficial layers decreased in the polylactide-based copolymer skin substitute and biosynthetic skin dressing-treated groups and initially increased in the untreated wounds and CCC groups on day 10; these values also decreased. The presented results demonstrate the complex changes in microcirculation in the course of healing partial-thickness wounds with different wound dressings and contribute to a better understanding of these wounds. However, based on the results of the study, a clear recommendation for a specific substitute is not yet possible.

Abstract 199: A 3-D Prevascularized Cardiac Muscle Construct for Analyzing hMSCs Engraftment and Differentiation Potential in vitro

Introduction: The most fundamental problem facing cardiac therapy, unlike vascular grafts and heart valves, is to repair and/or regenerate the damaged myocardium. Restricted myocardial regeneration after tissue damage and shortage of donor organs for cardiac transplantation are the major constraints of conventional therapies. The most daunting task in the field of cardiovascular tissue engineering is the creation and/or regeneration of an in vitro engineered cardiac muscle; tissue engineering is associated with two common underlying concerns for clinical applicability, viz., contractility and thickness. However, both the thickness and the contractility of the derived cardiac tissue are dependent on the vascularity of the construct. Hypothesis: Whether functioning vascularized cardiac tissue can be generated by the simultaneous interaction of cardiac myocytes, endothelial cells, and somatic stem cells, as would expect to occur during myocardial reparative/regenerative processes; by utilizing, viz., the embryo-derived embryonic cardiac myocytes (eCMs) and the human adipose-derived multipotent mesenchymal stem cells (hMSCs) on a three-dimensional (3-D) prevascularized collagen cell carrier (CCC) scaffold. Methods and Results: First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D CCC for 7 days under vasculogenic culture conditions, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Lastly, expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, not only were hMSCs able to couple electromechanically with developing eCMs, but also able to contribute to the developing vasculature as mural cells, respectively. Conclusions: Hence, our unique 3-D co-culture system provides us a reproducible and quintessential in vitro 3-D model of cardiomyogenesis, and a functional cardiac graft that can be utilized for personalized medicine.

Engineered cartilage regeneration from adipose tissue derived-mesenchymal stem cells: A morphomolecular study on osteoblast, chondrocyte and apoptosis evaluation

The poor self-repair capacity of cartilage tissue in degenerative conditions, such as osteoarthritis (OA), has prompted the development of a variety of therapeutic approaches, such as cellular therapies and tissue engineering based on the use of mesenchymal stem cells (MSCs). The aim of this study is to demonstrate, for the first time, that the chondrocytes differentiated from rat adipose tissue derived-MSCs (AMSCs), are able to constitute a morphologically and biochemically healthy hyaline cartilage after 6 weeks of culture on a Collagen Cell Carrier (CCC) scaffold. In this study we evaluated the expression of some osteoblasts (Runt-related transcription factor 2 (RUNX2) and osteocalcin), chondrocytes (collagen I, II and lubricin) and apoptosis (caspase-3) biomarkers in undifferentiated AMSCs, differentiated AMSCs in chondrocytes cultured in monolayer and AMSCs-derived chondrocytes seeded on CCC scaffolds, by different techniques such as immunohistochemistry, ELISA, Western blot and gene expression analyses. Our results showed the increased expression of collagen II and lubricin in AMSCs-derived chondrocytes cultured on CCC scaffolds, whereas the expression of collagen I, RUNX2, osteocalcin and caspase-3 resulted decreased, when compared to the controls. In conclusion, this innovative basic study could be a possible key for future therapeutic strategies for articular cartilage restoration through the use of CCC scaffolds, to reduce the morbidity from acute cartilage injuries and degenerative joint diseases.

289 - Collagen cell carrier for urethral reconstructive surgery: First results of a long-term minipig model

289 - Collagen cell carrier for urethral reconstructive surgery: First results of a long-term minipig model

A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct.

Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

Collagen cell carriers seeded with human urothelial cells for urethral reconstructive surgery: first results in a xenograft minipig model.

Urethral strictures are a common disease of the lower urinary tract in men. At present, the use of buccal mucosa is the method of choice for long or recurrent strictures. However, autologous tissue-engineered grafts are still under investigation for reconstructive urological surgery. The aim of this pilot study was to evaluate the use of human urothelial cells (HUC) seeded on bovine collagen type I-based cell carriers (CCC) in an animal model and to evaluate short-term outcome of the surgical procedure. Four male Göttingen minipigs were used with immunosuppression (cyclosporine A) for this pilot xenograft study. HUC obtained from human benign ureteral tissue were stained by PKH26 and seeded on a collagen cell carrier (CCC). Seven weeks after urethral stricture induction and protective vesicostomy, cell-seeded CCC was implanted in the urethra with HUC luminal and antiluminal, respectively. After two weeks animals were euthanized, urethrography and histological assessment were performed. Surgery was technically feasible in all minipigs. Stricture was radiologically established 7weeks after induction. CCC was visible after two weeks and showed good integration without signs of inflammation or rejection. In the final urethrography, no remaining stricture could be detected. Near porcine urothelium, PKH26-positive areas were found even if partially detached from CCC. Although diminished, immunofluorescence with pankeratin, CK20, E-cadherin and ZO-1 showed intact urothelium in several areas on and nearby CCC. Finally, this study demonstrates that the HUC-seeded CCC used as a xenograft in minipigs is technically feasible and shows promising results for further studies.

Biomechanical Skin Property Evaluation for Wounds Treated With Synthetic and Biosynthetic Wound Dressings and a Newly Developed Collagen Matrix During Healing of Superficial Skin Defects in a Rat Models.

There is a high prevalence of superficial wounds such as partial-thickness burns. Treatment of these wounds frequently includes temporary application of wound dressings. The aim of this study was to compare a newly developed collagen matrix with commonly used temporary skin dressings for treatment of partial-thickness skin defects. Through a skin dermatome, 42 standardized superficial skin defects were generated on the back of 28 adult male Lewis rats. The wounds were treated with a synthetic wound dressing (Suprathel, Polymedics Innovations Inc, Woodstock, GA) (n = 14), a biosynthetic skin dressing (Biobrane, Smith & Nephew, Hull, UK) (n = 14), or a newly developed bovine collagen matrix, Collagen Cell Carrier (Viscofan BioEngineering, Weinheim, Germany) (n = 14). Biomechanical properties of the skin were determined and compared every 10 days over a 3-month period of using the Cutometer MPA 580 (Courage + Khazaka Electronic GmbH, Cologne, Germany). As opposed to healthy skin, statistically significant differences were detected between days 10 and 30, and between days 60 and 80, for calculated elasticity (Ue), firmness of skin (R0), and overall elasticity (R8). After 3 months, no statistically significant differences in skin elasticity were detected between the different wound dressings. The presented results give an opportunity to compare the wound dressings used for treatment with respect to skin elasticity and reveal the potential of the bovine collagen matrix in the treatment of superficial skin defects; therefore the results facilitate further evaluation of collagen matrix in surgical applications and regenerative medicine.

Melanocytes from the outer root sheath of human hair and epidermal melanocytes display improved melanotic features in the niche provided by cGEL, oligomer-cross-linked gelatin-based hydrogel.

Non-invasively based cell treatments of depigmented skin disorders are largely limited by means of cell sampling as much as by their routes of application. Human melanocytes cultivated from the outer root sheath of hair follicle (HUMORS) are among the cell types that fit the non-invasive concept by being cultivated out of a minimal sample: hair root. Eventual implementation of HUMORS as a graft essentially depends on a choice of suitable biocompatible, biodegradable carrier that would mechanically and biologically support the cells as transient niche and facilitate their engraftment. Hence, the melanotic features of follicle-derived HUMORS and normal human epidermal melanocytes (NHEM) in engineered scaffolds based on collagen, the usual leading candidate for graft material for a variety of skin transplantation procedures were tested. Hydrogel named cGEL, an enzymatically degraded bovine gelatin chemically cross-linked with an oligomeric copolymer synthesized from pentaerythritol diacrylate monostearate (PEDAS), maleic anhydride (MA), and N-isopropylacrylamide (NiPAAm) or diacetone acrylamide (DAAm), was used. The cGEL provided a friendly three-dimensional (3D) cultivation environment for human melanocytes with increased melanin content of the 3D cultures in comparison to Collagen Cell Carrier® (CCC), a commercially available bovine decellularized collagen membrane, and electrospun polycaprolactone (PCL) matrices. One of the cGEL variants fostered not only a dramatic increase in melanin production but also a significant enhancement of melanotic gene PAX3, PMEL, TYR, and MITF expression in comparison to that of both CCC full-length collagen and PCL scaffolds, providing a clearly superior melanocyte niche that may be a suitable candidate for grafting carriers. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3115-3126, 2016.

Abstract 213: Evolving Challenges in Promoting Stem Cell Based Cardiovascular Repair and Regeneration

Introduction: Use of adult stem cells in the stimulation of mammalian cardiac muscle regeneration is in its infancy, and to date, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone-marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific cells, and also exhibit functional synchronization. As a result, this necessitates the development of an appropriate in vitro three-dimensional (3-D) model of cardiomyogenesis and prompts the development of a 3-D cardiac muscle construct for tissue engineering purposes, especially using the adult stem cells. Hypothesis: Functioning vascularized cardiac tissue can be generated by the interaction of human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human multipotent mesenchymal stem cells (hMSCs) on a 3-D prevascularized collagen cell carrier (CCC) scaffold. Methods and Results: In order to achieve the above aim, we have developed an in vitro 3-D functioning vascularized cardiac muscle construct using hiPSC-ECMs and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured on 3-D CCCs for 7 days under vasculogenic culture conditions, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of micro vessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed dramatic neo-angiogenesis and neo-cardiomyogenesis. Conclusions: Thus, our unique 3-D co-culture system provided us the apt in vitro functioning prevascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

Improvement of Split Skin Graft Quality Using a Newly Developed Collagen Scaffold as an Underlayment in Full Thickness Wounds in a Rat Model: Reply.

INTRODUCTION Frequently, full thickness skin defects were treated through split skin graft (SSG) transplantation. However, this is usually associated with a reduction of skin quality and scarring. In this context, a newly developed collagen cell carrier (CCC) has been evaluated as an underlayment in SSG transplantations in a rat model. MATERIALS AND METHODS Twenty-eight standardized full thickness skin defects were generated on the back of 28 adult male Lewis rats. The wounds were randomized and treated with SSG transplantation solitarily (n = 14) or SSG transplantation using CCC as an underlayment (n = 14). For skin quality analysis, a histological evaluation was performed 3 months postoperative in regard to epidermal cell count and epidermal and dermal thickness. RESULTS Wounds treated with SSG and CCC demonstrated a thicker epidermis and significantly higher epidermal cell count compared to SSG solitarily. At the time of evaluation, epidermal thickness of SSG and CCC was comparable to untreated, healthy skin (no statistically significant differences). DISCUSSION As split skin grafting is frequently associated with skin quality reduction, the presented results reveal the beneficial effects of CCC in terms of skin graft quality improvement. This may offer unique opportunities in wound management and encourages further evaluation of CCC in surgical applications and regenerative medicine.

Evaluation of commonly used temporary skin dressings and a newly developed collagen matrix for treatment of superficial wounds.

In the authors' experience, extended superficial-thickness wounds often were treated with the application of synthetic temporary wound dressings. There is a broad range of expensive products available in this market. The aim of this study was to compare commonly used temporary skin dressings with a newly developed collagen matrix. A total of 56 standardized superficial skin defects were generated on the back of 28 adult male Lewis rats using a skin dermatome. The wounds were treated with Biobrane (Smith & Nephew, St Petersburg, Florida) (n = 14), Suprathel (PolyMedics, Denkendorf, Germany) (n = 14), and newly developed Collagen Cell Carrier (Naturin Viscofan, Weinheim, Germany) (CCC; n = 14). Histological evaluation was performed at 3 months' postoperative in regard to quality of newly formed skin. In regard to epidermal cell count and thickness of skin, Biobrane and Suprathel demonstrated comparable values, whereas CCC demonstrated higher epidermal cell count and thicker neoepidermis. The epidermal thickness of CCC was comparable to untreated, healthy skin. The presented results reveal the potential of CCC in the treatment of superficial skin defects and encourages further evaluation of CCC in surgical applications and regenerative medicine.

A Bovine Collagen Type I-Based Biodegradable Matrix as a Carrier for Tissue-Engineered Urothelium

Engineering of lower urinary tract tissue suitable for reconstructive surgery requires biomaterials as cell carriers, particularly in patients for whom autologous grafts are not available. Matrices should support growth, improve mechanical stability, feature excellent biocompatibility, and fully degrade without signs of scarring at the implantation side. In this study, a new bovine collagen type I-based biodegradable non-cross linked matrix was investigated for its suitability as a carrier for porcine and human urothelial cells in vitro. Initial cell adherence, metabolic activity, and proliferation behaviour of cells isolated from tissue biopsies were analyzed. Constructs were characterized immunohistologically in comparison with matrix-free cell sheets established on plastic surface (=controls). Even for high-density seeding, adherence on collagen cell carrier (CCC) was excellent. Metabolic activity and proliferation of stratifying porcine and human urothelial cells cultured on CCC were comparable to that of controls. Immunofluorescence analysis confirmed epithelial phenotype, cell-cell junction formation and ongoing differentiation of the multilayered urothelium on CCC. This study proved CCC as a suitable carrier for urothelial cells for the future aim for urethral reconstruction.

In Vivo Biocompatibility Testing of a Collagen Cell Carrier Seeded with Human Urothelial Cells in Rats

Tissue-engineered, matrix-stabilized autologous urothelium is a new option for urethral reconstruction, particularly for patients for whom other autologous grafts are not available. In vitro engineering of urothelial tissue requires biomaterials as cell carriers that increase the stability of cell-based implants. The aim of this study was to investigate a new highly standardized, industrially manufactured bovine collagen type I-based cell carrier (CCC) for its suitability as a carrier matrix for human urothelial cells (HUC). As an in vivo biocompatibility test the behaviour and degradation of these implants was to be proven in a nude rat model. Expanded HUC from tissue biopsies were phenotypically analysed by immuno¬cyto¬chemistry and seeded onto CCC. For in vivo application, CCC was seeded with PKH26-labelled HUC in high density and constructs were implanted onto the rectus abdominis muscle of nude rats. Integration, cell survival, and degradation of the urothelium-matrix-implants were studied after 1, 2, and 4 weeks. Immunohistological characterization of multilayered urothelium-matrix-constructs was performed for AE1/AE3 and p63 (epithelial phenotype), CK20 (differentiation), and E-Cadherin and ZO-1 (junction formation). Immunocytochemical staining showed urothelial character of the isolated cells and the absence of fibroblasts and muscle cells. In twelve nude rats, urothelium-matrix-implants integrated well into the host tissue where no inflammation was observed. Immuno¬fluorescence analysis confirmed epithelial phenotype, adherence and tight junction formation homogeneous multilayer formation of stratified HUC cultures as well as urothelial phenotype and differentiation in vitro the CCC-matrix additionally revealed promising biocompatibility investigated in a nude rat model. Thus, this study demonstrates excellent suitability of CCC as a matrix for urothelial cells and recommends its use in a large animal model with regard to clinical application.

In vivobiocompatibility and biodegradation of a novel thin and mechanically stable collagen scaffold

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable, and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabeled for CD68 and stained by hematoxylin-eosin and Masson-Goldner's trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long-term vascularization and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption, and replacement by autologous tissue. The in vivo biocompatibility and degradation behavior encourage for further evaluation of CCC in surgical applications and regenerative medicine.

791 Seeding a biodegradable collagen cell carrier and its use for urethroplasty in a urethral stricture minipig model

INTRODUCTION AND OBJECTIVES: For the surgical treatment of long and complicated urethral strictures, bioartificial matrixstabilized urothelium offers an innovative therapeutic option for urethral reconstruction. Collagen matrices increase the stability of cell-based implants and promote cell viability and proliferation of urothelial cells. In this study a Collagen Cell Carrier (CCC) seeded with autologous urothelial cells was implanted in minipigs after induction of a urethral stricture. METHODS: Six minipigs underwent surgical procedures for urethral stricture induction by circular thermocoagulation and vesicostomy to guarantee urinary diversion together with bladder tissue harvest to isolate autologous porcine urothelial cells(PUC). PUC were expanded, labelled by PKH26, and seeded onto the CCC in high density. Stratification was induced in confluent PUC cultures. Constructs were applied after seven days. Three weeks after stricture induction, minipigs were treated with autologous urothelium-matrixtransplants for urethroplasty at stricture side. After one, two, and four weeks, animals were sacrificed and urethral tissues were histologically examined for integration and survival of the seeded CCC. In vivo phenotype of multilayered urothelium-matrix-constructs were characterized via immunofluorescence staining of AE1/AE3, CK20, p63, Ecadherin, and ZO-1. RESULTS: All animals tolerated the required interventions and recovered quickly. The seeded CCC displayed excellent stability characteristics and suturability when manipulated and applied in the minipigs. Transplanted cells could be detected via positive PKH26 fluorescence in all minipigs, although cells were partly shifted from the CCC. Urothelium-matrix transplants integrated very well into the host tissue without any sign of visible inflammation. Throughout the complete experimental period, none of the animals showed symptoms of rejection or impairments. Blood counts did not reveal major aberrations. Immunofluorescence analysis confirmed epithelial phenotype, junction formation and ongoing differentiation two weeks post-op. CONCLUSIONS: Urethroplasty using autologous urothelial cells seeded on CCC was feasible in minipigs after induction of urethral stricture. This study demonstrates excellent suitability of the cellseeded CCC for urologic reconstruction purposes and recommends its use in clinical application.

Improvement of Skin Quality Using a New Collagen Scaffold in Acute Burns and Reconstructive Surgery: An In Vivo Evaluation of Split Skin Graft Transplantation in a Rat Model

Split-thickness skin grafting is often associated with poor skin quality. In this context, a new collagen cell carrier (CCC), as an underlayment in split skin graft (SSG) transplantation for covering tissue defects in rats, has been evaluated as an improving agent. Twenty-eight full-thickness wounds were covered with SSGs, applying the CCC as an underlayment in 14 rats and using SSG transplantation alone in control group. Postgraft skin areas were assessed using an instrument that measures mechanical properties of skin. Three parameters were considered for skin elasticity analysis: total skin deviation (R0), gross elasticity (R2) and viscoelasticity (R8). Measurements were performed every 10 days for 80 days after grafting. Biopsies were taken subsequently for histologic evaluation. The results demonstrated significantly superior elasticity values in CCC-supplemented SSGs in terms of gross elasticity and viscoelasticity (R2/R8) starting from day 60 after grafting to the end of the measuring period. There was no histologic evidence of inflammation, adverse host tissue reaction, or scar tissue formation. Split skin grafting is associated with poor dermal quality, but CCC may offer unique opportunities in complex wound management in terms of skin graft quality improvement.

Evaluation of a Thin and Mechanically Stable Collagen Cell Carrier

The biological function of adherent cell populations strongly depends on the physical and biochemical properties of extracellular matrix molecules. Therefore, numerous biocompatible cell carriers have been developed to specifically influence cell attachment, proliferation, cellular differentiation, and tissue formation for diverse cell culture applications and cell-based therapies. In the present study, we evaluated the mechanical and the cell biological properties of a novel, thin, and planar collagen scaffold. The cell carrier is based on fibrillar bovine collagen type I and exhibits a low material thickness coupled with a high mechanical stability as measured by tensile tests. The influence of this new biomaterial on cell viability, proliferation, and cell differentiation was analyzed using 5-bromo-2-deoxyuridine (BrdU) proliferation assay, immunocytochemistry, water-soluble tetrazolium salt-1 assay (WST-1), live cell imaging, and electron microscopy. Cell culture experiments with the human osteosarcoma cell line Saos-2, human mesenchymal stem cells, and rodent cardiomyocytes demonstrated the in vitro biocompatibility of this chemically noncrosslinked scaffold. Both the mechanical characteristics and the in vitro biocompatibility of this collagen type I carrier facilitate the engineering of thin transferable tissue constructs and offer new possibilities in the fields of cell culture techniques, tissue engineering, and regenerative medicine.