3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells.

Machado CB, Ventura JM, Lemos AF, Ferreira JM, Leite MF, Goes AM.

Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Brazil.

Abstract
A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

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In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells.

Wang Y, Kim UJ, Blasioli DJ, Kim HJ, Kaplan DL.

Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, USA.

Abstract
Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.

Keywords: Mesenchymal stem cell; MSC; Cartilage tissue engineering; Aqueous-derived silk scaffold\

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In vivo biocompatibility and mechanical properties of porous zein scaffolds.

Wang HJ, Gong SJ, Lin ZX, Fu JX, Xue ST, Huang JC, Wang JY.

College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China.

Abstract
In our previous study, a three-dimensional zein porous scaffold with a compressive Young's modulus of up to 86.6+/-19.9 MPa and a compressive strength of up to 11.8+/-1.7 MPa was prepared, and was suitable for culture of mesenchymal stem cells (MSCs) in vitro. In this study, we examined its tissue compatibility in a rabbit subcutaneous implantation model; histological analysis revealed a good tissue response and degradability. To improve its mechanical property (especially the brittleness), the scaffolds were prepared using the club-shaped mannitol as the porogen, and stearic acid or oleic acid was added. The scaffolds obtained had an interconnected tubular pore structure, 100-380 microm in pore size, and about 80% porosity. The maximum values of the compressive strength and modulus, the tensile strength and modulus, and the flexural strength and modulus were obtained at the lowest porosity, reaching 51.81+/-8.70 and 563.8+/-23.4 MPa; 3.91+/-0.86 and 751.63+/-58.85 MPa; and 17.71+/-3.02 and 514.39+/-19.02 MPa, respectively. Addition of 15% stearic acid or 20% oleic acid did not affect the proliferation and osteogenic differentiation of MSCs, and a successful improvement of mechanical properties, especially the brittleness of the zein scaffold could be achieved.

Keywords: Scaffold; Zein; Fatty acid; Brittleness; Mechanical properties; Subcutaneous implantation

Source: http://www.sciencedirect.com/science/article/pii/S0142961207004164

Evaluation of the zein/inorganics composite on biocompatibility and osteoblastic differentiation.

Qu ZH, Wang HJ, Tang TT, Zhang XL, Wang JY, Dai KR.

Orthopaedic Cellular and Molecular Biology Laboratory, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai JiaoTong University School of Medicine, PR China.

Abstract
We have previously studied the biocompatibility and mechanical properties of porous zein scaffolds. We based the study on the concept that composite scaffold materials, especially when combined with inorganic materials, are more suited to the mechanical and physiological demands of the host tissue than individual scaffold materials. We investigated the effect of zein/inorganic composite on the physical and biological properties of porous zein scaffolds, which were fabricated by salt-leaching. The composite was prepared by immersion in simulated body fluid. The hydroxyapatite (HA)-coated zein scaffold had the same porosity as the zein scaffold (over 75%). Using scanning electron microscopy, it was established that the morphology of pores located on the surface and within the porous scaffolds showed equally good pore interconnectivity with zein. However, the compressive Young's modulus decreased from 240.1+/-96.8 to 34.4+/-12.6MPa, and compressive strength decreased from 7.8+/-1.2 to 4.2+/-0.8MPa. From the in vitro test with human bone marrow stroma cells, the osteoblastic differentiation on the surface of the HA-coated zein scaffold was increased, as expressed by the alkaline phosphatase activity and reverse transcription-polymerase chain reaction analysis for marker genes. From both the mechanical and biological evaluations, the HA-coated zein scaffold was found to be the optimal biomaterial for bone tissue engineering.

Keywords: Scaffold; Zein; Hydroxyapatite; Mechanical properties; Osteogenic

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Mechanical properties and in vitro biocompatibility of porous zein scaffolds.

Gong S, Wang H, Sun Q, Xue ST, Wang JY.

College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, PR China.

Abstract
A porous scaffold utilizing hydrophobic protein zein was prepared by the salt-leaching method for tissue engineering. The scaffolds possessed a total porosity of 75.3-79.0%, compressive Young's modulus of (28.2+/-6.7)MPa-(86.6+/-19.9)MPa and compressive strength of (2.5+/-1.2)MPa-(11.8+/-1.7)MPa, the percentage degradation of 36% using collagenase and 89% using pepsin during 14 days incubation in vitro. The morphology of pores located on the surface and within the porous scaffolds showed good pore interconnectivity by scanning electron microscopy (SEM). Rat mesebchymal stem cells (MSCs) could adhere, grow, proliferate and differentiate toward osteoblasts on porous zein scaffold. With the action of dexamethasone, the cells showed a relative higher activity of alkaline phosphatase (ALP) and a higher proliferating activity (p<0.05) than those of MSCs without dexamethasone.

Keywords: Scaffold; Zein; Mechanical properties; Porosity; Degradation; MSCs

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Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering.

Wu C, Zhou Y, Lin C, Chang J, Xiao Y.

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People's Republic of China.

Abstract
To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which can stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there has been no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate osteogenic/cementogenic differentiation of PDLCs in a tissue-engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nanopore volume and nanopore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr(2+) ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in a simulated body fluid environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue-engineering applications.
Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Periodontal tissue engineering; Strontium; Mesoporous bioglass; Osteogenic/cementogenic differentiation; Periodontal ligament cells

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Effect of shuanghuangbu on the adhesion growth and proliferation of human periodontal ligament cells on the zein stent

[Article in Chinese]
Xu YZ, Li N, Liu J.

Department of Stomatology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011.

Abstract

OBJECTIVE:
To study the effect of Shuanghuangbu on the adhesion growth and proliferation of human periodontal ligament cells (HPDLCs) on the zein scaffold, and to study the feasibility of Shuanghuangbu zein used in the periodontal tissue engineering as a compound scaffold material.
METHODS:
HPDLCs were cultured in vitro using the explants adherent method. Shuanghuangbu Decoction was prepared using water extraction and alcohol precipitation method. Zein scaffold was prepared using solvent casting/corpuscle leaching method, which was dispensed into Shuanghuangbu culture fluid at the concentration of 50, 100, 150, 200, 500, 1000 microg/mL, respectively. Shuanghuangbu of different concentrations groups, the zein scaffold leaching liquid group, and the cell control group were also set up. The effect of each group on the proliferation of HPDLCs was detected by MTT. According to results of MTT, the best concentration of Shuanghuangbu (100 microg/mL) was selected as the experiment group. Ten percent FBS of DMEM culture fluid was taken as the control group. The effect of Shuanghuangbu on the morphology of HPDLCs on the zein scaffold was observed using inverted phase contrast microscope and scanning electron microscope.
RESULTS:
Compared with the zein scaffold leaching liquid group and the cell control group, the proliferation of HPDLCs could be promoted by different concentrations of Shuanghuangbu culture fluid, with the most obvious effect shown by Shuanghuangbu at 100 microg/mL (P<0.01). There was no obvious difference between the zein scaffold leaching liquid group and the cell control group (P>0.05), but their cell proliferation rates were positive values. The morphological results demonstrated that HPDLCs successfully grew on the zein scaffold. The number of HPDLCs under the action of Shuanghuangbu was more and the morphous was better.
CONCLUSIONS:
Shuanghuangbu could promote the adhesion growth and proliferation of HPDLCs on the zein scaffold. Shuang- huangbu zein was feasible to be used in the periodontal tissue engineering as a compound scaffold materia.

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Zhongguo Zhong Xi Yi Jie He Za Zhi. 2011 Jul;31(7):932-6

Growth factor release from tissue engineering scaffolds.

Whitaker MJ, Quirk RA, Howdle SM, Shakesheff KM.

School of Pharmaceutical Sciences, The University of Nottingham, UK.

Abstract
Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

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J Pharm Pharmacol. 2001 Nov;53(11):1427-37.

Synthetic extracellular matrices for tissue engineering and regeneration.

Silva EA, Mooney DJ.

Division of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA.

Abstract
The need for replacement tissues or organs requires a tissue supply that cannot be satisfied by the donor supply. The tissue engineering and regeneration field is focused on the development of biological tissue and organ substitutes and may provide functional tissues to restore, maintain, or improve tissue formation. This field is already providing new therapeutic options to bypass the limitations of organ?tissue transplantation and will likely increase in medical importance in the future. This interdisciplinary field accommodates principles of life sciences and engineering and encompasses three major strategies. The first, guided tissue regeneration, relies on synthetic matrices that are conductive to host cells populating a tissue defect site and reforming the lost tissue. The second approach, inductive strategy, involves the delivery of growth factors, typically using drug delivery strategies, which are targeted to specific cell populations in the tissues surrounding the tissue defect. In the third approach, specific cell populations, typically multiplied in culture, are directly delivered to the site at which one desires to create a new tissue or organ. In all of these approaches, the knowledge acquired from developmental studies often serves as a template for the tissue engineering approach for a specific tissue or organ. This article overviews the development of synthetic extracellular matrices (ECMs) for use in tissue engineering that aim to mimic functions of the native ECM of developing and regenerating tissues. In addition to the potential therapeutic uses of these materials, they also provide model systems for basic studies that may shed light on developmental processes.

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Bioabsorbable polymer scaffolds for tissue engineering capable of sustained growth factor delivery.

Sheridan MH, Shea LD, Peters MC, Mooney DJ.

Department of Biological & Materials Sciences, University of Michigan, Ann Arbor, MI 48109-1078, USA.

Abstract
Engineering new tissues utilizing cell transplantation on biodegradable polymer matrices is an attractive approach to treat patients suffering from the loss or dysfunction of a number of tissues and organs. The matrices must maintain structural integrity during the process of tissue formation, and promote the vascularization of the developing tissue. A number of molecules (angiogenic factors) have been identified that promote the formation of new vascular beds from endothelial cells present within tissues, and the localized, controlled delivery of these factors from a matrix may allow an enhanced vascularization of engineered tissues. We have developed a gas foaming polymer processing approach that allows the fabrication of three-dimensional porous matrices from bioabsorbable materials (e.g., copolymers of lactide and glycolide [PLG]) without the use of organic solvents or high temperatures. The effects of several processing parameters (e.g., gas type, polymer composition and molecular weight) on the process were studied. Several gases (CO(2), N(2), He) were utilized in the fabrication process, but only CO(2) resulted in the formation of highly porous, structurally intact matrices. Crystalline polymers (polylactide and polyglycolide) did not form porous matrices, while amorphous copolymers (50:50, 75:25, and 85:15 ratio of lactide:glycolide) foamed to yield matrices with porosity up to 95%. The mechanical properties of matrices were also regulated by the choice of PLG composition and molecular weight. Angiogenic factors (e.g., vascular endothelial growth factor) were subsequently incorporated into matrices during the fabrication process, and released in a controlled manner. Importantly, the released growth factor retains over 90% of its bioactivity. In summary, a promising system for the incorporation and delivery of angiogenic factors from three-dimensional, biodegradable polymer matrices has been developed, and the fabrication process allows incorporation under mild conditions.

Keywords: Angiogenesis; Poly (lactide-co-glycolide); Alginate; Tissue engineering; Vascular endothelial growth factor

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