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Potential use of collagen-chitosan-hyaluronan tri-copolymer scaffold for cartilage tissue engineering.

A three-dimensional biodegradable porous scaffold plays a vital role in a tissue engineering approach. Collagen, chitosan and hyaluronan (HA) are natural extracellular matrix (ECM) or similarity, and may provide appropriate environment for the generation of cartilage-like tissue. In this study, we prepared a collagen/chitosan/HA tri-copolymer porous scaffold by freezing and lyophilization to evaluate physico-chemical properties of the tri-copolymer scaffold and its capacity to sustain chondrocytes proliferation and differentiation in vitro. The results show that the mechanical strength, the resistance to enzymatic degradation, and the waterblinding capacity were improved when chitosan and hyaluronan were incorporated into a collagen scaffold. After 21 days of culture, the porous scaffold had been surfaced with cartilaginous tissue. DNA and glycosaminoglycan (GAG) contents were significantly higher during culture periods in collagen/ chitosan/hyaluronan matrix compared to collagen alone matrix, and most seeded cells preserved the chondrocytic phenotype during culture within the scaffold. The collagen/chitosan/hyaluronan tri-copolymer scaffold has potential applications in a cartilage tissue engineering scaffold field.

Animals↗

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation.

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.

Animals↗

Nanoindentation on porous bioceramic scaffolds for bone tissue engineering.

We report nanoindentation mechanical properties measurements on porous ceramic scaffolds made for tissue engineering applications. The scaffolds have been made from tricalcium phosphate (TCP), hydroxyapatite (HA) nanopowder and mixed powders of HA (50 wt%) and TCP (50 wt%) using the polyurethane sponge method, which produces open porous ceramic scaffolds through replication of a porous polymer template. The scaffolds prepared by this method have a controllable pore size and interconnected pore structure. The crystal structures and morphology of porous scaffolds were determined by X-ray diffraction (XRD) and atomic force microscopy (AFM) respectively. Nanoindentation measurements to a depth of 600 nm showed a Young's modulus value of 10.3 GPa for HA+TCP composite scaffolds and 1.5 GPa for TCP scaffolds. The hardness values were 240 MPa for HA+TCP composites and 21 MPa for TCP sample respectively. The results showed that the mechanical properties of the biodegradable scaffolds can be considerably enhanced with the addition of HA while maintaining the interconnected open pores and pore geometry desirable for bone tissue engineering.

Biocompatible Materials↗

[From wound healing to modern tissue engineering of the skin. A historical review on early techniques of cell and tissue culture].

The early precursors of modern techniques of keratinocyte transplantation and tissue engineering of the skin are reviewed, starting with histological research into the processes of wound healing and of skin transplantation in the 19th century and continuing with the introduction of cell and tissue culture techniques in early 20th century medicine. The later experimental and clinical application of this research in dermatology and plastic surgery is also explored.

Animals↗

[Induction of transforming growth factor-beta 1 and dentin non-collagen proteins on tissue engineering pulp].

OBJECTIVE: To study the influence of transforming growth factor-beta 1 (TGF-beta 1), dentin non-collagen proteins (dNCPs) and their complex on tissue engineering pulp system. METHODS: Collagen I and dentin powder were used to construct the system of pulp cells in 3-dimensional culture, dentin powder was added in the gel. The tissue engineering pulp were divided TGF-beta 1 group, dNCPs group, TGF-beta 1/dNCPs group and control group. After 3, 6 and 14 days, the appearance and the differentiation of pulp cells were observed by HE staining and immunohistochemical staining respectively. RESULTS: Collagen I could form netted collagen gel construction. Growing condition of pulp cells in gel was similar to that of pulp cells in vivo. After the TGF-beta 1 and dNCPs were added, the pulp cells had some characteristics of odontoblasts and had unilateral cell process after culture 6 days. Pulp cells arranged with parallel columnar and form dentin-pulp-like complex after 14 days. Immunohistochemical staining showed dentin salivary protein (DSP) began to express in some cells. The number of positive cell was most in the TGF-beta 1 group. No positive cells were detected in the control group. CONCLUSION: The transforming growth factor-beta 1 and non-collagen proteins can stimulate the pulp cells to transform into odontoblasts to some extent, which promote the formation of tissue engineering pulp.

Animals↗

Cell transplantation for heart failure, and tissue engineering of cardiovascular structures.

The emergence of selective cell transplantation and tissue engineering has opened up new alternatives for the treatment of heart failure. These involve the use of stem and progenitor cells, as well as new biodegradable polymer scaffolds. The last two decades have seen a dramatic increase in our knowledge of how to fabricate a wide array of tissues, including cardiovascular structures. This article reviews current trends in selective cell transplantation and tissue engineering, and summarises recent achievements and remaining questions in constructing functional cardiac tissue.

Journal Article↗

Viability evaluation of engineered tissues.

Biohybrid artificial organs encompass all devices capable of substituting for an organ or tissue function and are fabricated from both synthetic materials and living cells. The viability of engineered tissue could be related to the viability of implanted cells. The system of viability assay for mammalian cell culture can be applied to the determination of cell viability for engineered tissue. This review explores various methods of cell viability assay which can be applied to the viability evaluation of engineered tissue. The major criteria employed in viability assays include survival and growth in tissue culture, functional assay, metabolite incorporation, structural altercation, and membrane integrity. Each viability assay method is based on different definitions of cell viability, and has inherent advantages and disadvantages. In order to be able to assess the viability of cells with one assay method, it is desirable to compare the viability measurements from various assays derived from different criteria.

Animals↗

Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine.

BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer tubes. This study investigated methods of stimulation for optimizing neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes and implanted into the omentum of adult Lewis rats in the following groups: (1) the control group (group C), implantation alone (n=9); (2) the small bowel resection (SBr) group, after 75% SBr (n=9); (3) the portacaval shunt (PCS) group, after PCS (n=8); and (4) the partial hepatectomy (PH) group, after 75% PH (n=8). Neointestinal cyst size was recorded using ultrasonography. Constructs were harvested at 10 weeks and were examined using histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program (NIH Image, version 1.59). RESULTS: Cyst development was noted in all animals. Cyst lengths and diameters were significantly larger in the SBr group at 7 and 10 weeks compared with the other three groups (P<0.05; analysis of variance [ANOVA], Fisher's protected least significant difference). Histology revealed a well-vascularized tissue with a neomucosa lining the lumen with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated a significantly greater villus number, height, area, and mucosal surface in the SBr group compared with the other three groups and a significantly greater crypt number and area in the PCS group compared with group C (P<0.05; ANOVA, Fisher's protected least significant difference). CONCLUSIONS: Intestinal epithelial organoid units transplanted on porous biodegradable polymer tubes can successfully vascularize, survive, and regenerate into complex tissue resembling small intestine. SBr and, to a lesser extent, PCS provide significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered small intestine.

Animals↗

Rational design of hydrogels for tissue engineering: impact of physical factors on cell behavior.

When designing suitable biomaterials for tissue-engineering applications, biological and chemical parameters are frequently taken into account, while the equally important physical design variables have often been neglected. For a rational design of biomaterials, however, all variables influencing cell function and tissue morphogenesis have to be considered. This review will stress the development of cross-linked hydrogels and outline the impact of their physical properties on cell function and tissue morphogenesis. In the first part, the principles of cellular mechanosensitivity, as well as the influence of substrate mechanics on cell behavior, will be discussed. Afterwards, methods to characterize the mechanical properties of biomaterials will be presented. The subsequent chapters will address hydrogels that allow for the control of their physical qualities followed by a discussion of their use in tissue-engineering applications.

Biocompatible Materials↗

Poly(phosphoester) ionomers as tissue-engineering scaffolds.

Regenerative medicine requires scaffolds of divergent physicochemical properties for different tissue-engineering applications. To this end, a series of biodegradable poly(phosphoester) ionomers of the general composition [p(BHET-EOP-HOP/TC)] was synthesized, with BHET(bis-hydroxyl ethylene phosphate):EOP(ethylene phosphate):HOP(free phosphate) ratios of 60:20:20, 70:10:20, and 75:5:20, respectively. The 60/20/20 ionomer possessed the best tensile properties, exhibiting an average tensile modulus of 68 MPa and strain at break of 31%. Calcium treatment of the ionomer films led to significantly higher hardness and elastic moduli as measured by indentation. Calcium binding was evident from the increase in glass transition and melting temperatures, and a shift in the P-->O absorption in the FTIR spectrum. Stable N-hydroxysuccinimide ester (NHS) of the ionomers could be synthesized to facilitate derivatization, as demonstrated by conjugation of GRGDS in this study. The polymers conjugated with NHS were hydrolyzed in a biphasic mode, with a fast initial phase occurring in the first few hours, followed by a slower phase in the next few days. These ionomers represent a novel class of biomaterials with readily controllable physical and chemical attributes for tissue engineering.

Biocompatible Materials↗

[Experimental study on repair of critical-sized cranial defect by tissue engineered bone].

OBJECTIVE: To evaluate repair of critical-sized cranial defect with tissue engineered bone fabricated by coral, bone mesenchymal stem cells (MSCs) and sustainedly released recombinant human bone morphogenetic protein 2 (rhBMP-2) by collagen. METHODS: Three scaffolds of rhBMP-2 + coral, collagen + rhBMP-2 + coral and MSCs+collagen+rhBMP-2+coral were fabricated. Forty New Zealand rabbits were made the models of critical-sized defects and divided into 5 groups according to different implants: group I, auto-ilium; group II, coral; group III, rhBMP-2+coral; group IV, collagen+rhBMP-2+coral; and group V , MSCs+collagen+rhBMP-2+coral. Repair of bone defect was evaluated after 8 and 16 weeks of implantation by gross observation, X-ray, HE staining and Masson's trichrome staining. RESULTS: Repair of bone defect in group V was similar to that in group I, and was better than that in group IV; and group III was worse. The gross appearance showed that defect region filled with bony tissue which had similar strength to adjacent bone and formed bone union with surrounding bone. The X-ray result displayed high radiopacity (80.45% +/- 2.52% in the 16th week). Histological observation showed new lamellar bone tissue and with few pore blank area. However, only transparent fibrous tissue filled the defect in group II. CONCLUSION: Collagen may be a suitable sustained release system for rhBMP-2. And MSCs may have important effect on enhancing repair of bone defect. Tissue engineered bone fabricated by MSCs+collagen+rhBMP-2+coral may be a useful material for bone defect repair.

Animals↗

Bioresponsive phosphoester hydrogels for bone tissue engineering.

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

Alkaline Phosphatase↗

Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin-hydroxyapatite for tissue engineering scaffolds.

Collagen-derived gelatin/hydroxyapatite (HA) nanocomposites were biomimetically synthesized for hard tissue engineering scaffold. In vitro osteoblastic cellular responses to the nanocomposites were assessed in comparison with those conventionally mixed gelatin-HA composites. A three-dimensional culture method involving floating cells in a culture medium was introduced to assist in the initial attachment of the cells to the scaffolds, and the proliferation and differentiation behaviors of the cells were examined. The osteoblastic MG63 cells attached to the nanocomposites to a significantly higher degree and subsequently proliferated more. The alkaline phosphatase (ALP) activity and osteocalcin produced by the cells were significantly higher on the nanocomposite scaffolds than on the conventional composite scaffolds. These improved cellular responses on the nanocomposites are considered to result from the increased ionic release and serum protein adsorption on the nanocomposites, which was derived from the different structural and morphological characteristics, i.e., the nanocomposite scaffolds retained less-crystallized and smaller-sized apatite crystals and a more well-developed pore configuration than the conventional ones. Based on these findings, the biomimetically synthesized nanocomposite scaffolds are believed to be potentially useful in hard tissue regeneration and tissue engineering fields.

Alkaline Phosphatase↗

Porous chitosan tubular scaffolds with knitted outer wall and controllable inner structure for nerve tissue engineering.

In this study, a novel method was developed to create porous tubular scaffolds with desirable mechanical properties and controllable inner structure from chitosan, for nerve tissue engineering. Chitosan fiber-based yarns were first used to create porous hollow tubes, which served as the outer wall of the scaffolds, through an industrial knitting process. Then, an innovative molding technique was developed and used to produce inner matrices with multiple axially oriented macrochannels and radially interconnected micropores. Acupuncture needles were used as mandrels during molding to improve the safety and controllability of the process. In vitro characterization demonstrated that the scaffolds possessed suitable mechanical strength, porosity, swelling, and biodegradability for applications in nerve tissue engineering. In vitro cell culture experiments showed that differentiated Neuro-2a cells grew along the oriented macrochannels and the interconnected micropores were beneficial for nutrient diffusion and cell ingrowth to the scaffold's interior. Collectively, the well-defined architectural features in addition to the desirable mechanical and biological properties of the scaffolds make them promising for nerve tissue engineering.

Animals↗

Genetic marking with the DeltaLNGFR-gene for tracing goat cells in bone tissue engineering.

The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (DeltaLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40-60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with DeltaLNGFR is applicable to trace goat BMSC's in bone tissue engineering research.

Animals↗

Tissue engineering and dentistry.

This article assesses the current state of the art of tissue engineering and its applications in dentistry, especially in the reconstruction and repair of orofacial tissues, including bone and oral mucosa, for the restoration of function and improved cosmetics. The article discusses at length the tissue engineering of bone and cartilage, including guided tissue techniques, in which some of the most interesting advances in the field are being made.

Biocompatible Materials↗

Tissue-engineered human vascular media produced in vitro by the self-assembly approach present functional properties similar to those of their native blood vessels.

We have developed a tissue-engineering approach for the production of a completely biological blood vessel from cultured human cells. In the present study, we took advantage of this tissue-engineering method to demonstrate that it can be used to reproduce the subtle differences in the expression of receptors present on the media of native human blood vessels. Indeed, a small percentage (3 of 18) of native human umbilical cord veins (HUCVs) responded to endothelin, the most powerful vasopressor agent known to date, via both endothelin A (ET(A)) and endothelin B (ET(B)) receptor activation. In contrast, most HUCVs tested responded to ET via ET(A) receptor activation only. Tissue-engineered vascular media (TEVM) were next reconstructed by using vascular smooth muscle cells (VSMCs) isolated and cultured from HUCVs expressing both ET(A) and ET(B) receptors to determine the functional integrity of our TEVM model. The reconstructed TEVM presents an endothelin response similar to that of respective HUCVs from which VSMCs were isolated. Reverse transcriptase polymerase chain reaction on TEVM reconstructed in vitro correlated these vasocontractile profiles by showing the presence of messenger RNA for both ET(A) and ET(B) receptors. Taken together with recently published results on TEVM expressing only ET(A) receptor, these results show that our reconstructed TEVM present a similar ET response profile as the blood vessel from which the VSMCs were isolated and cultured. These findings indicate that subtle differences, such as receptor expression, are preserved in the reconstructed tissue. Therefore, our TEVM offers a valuable human in vitro model with which to study the functionality of human blood vessels, such as their vasoactive response, or to perform pharmacologic studies.

Blood Vessels↗

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix.

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.

Burns↗