Manipulations in hydrogel degradation behavior enhance osteoblast function and mineralized tissue formation

Hydrogels were prepared by copolymerizing a degradable macromer, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) endcapped with methacrylate groups (PEG-LA-DM), with a nondegradable macromer, poly(ethylene glycol) dimethacrylate (PEGDM). Copolymer networks consisted of 100:0, 83:17, 67:33, and 50:50 PEGDM:PEG-LA-DM mass%, essentially creating scaffolds that exhibit 0, 17, 33, and 50% degradation over the time course of the experiment. Osteoblasts were photoencapsulated in these copolymer hydrogels and cultured for 3 weeks in vitro. Metabolic activity, proliferation, and alkaline phosphatase production were enhanced by an increase PEG-LADM content and corresponding degradation. Gene expression of the cultured osteoblasts, normalized to beta-actin, was analyzed, and osteopontin and collagen type I gene expression increased with degradation. Finally, as a measure of mineralized tissue formation, calcium and phosphate deposition was analyzed biochemically and histologically. Mineralization increased with increasing concentration of PEG-LA-DM and biochemically resembled that of hydroxyapatite.     

A hydrogel derived from decellularized dermal extracellular matrix

The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in?vitro and in?vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In?vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in?vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior.     

脱细胞基质水凝胶在再生医学中的研究进展

脱细胞基质(dECM)由于其高仿生性和优异的生物相容性,已被广泛用作再生医学的支架材料。水凝胶(hydrogel)作为一种高含水量、可控流动性的功能高分子材料,非常适合用于临床中的部分微创手术。近年来,随着水凝胶理论和技术的快速发展,dECM水凝胶逐渐成为再生医学领域的研究热点。本文从dECM水凝胶的制备及其临床前应用两个方面对近年来的相关研究进行综述,并展望其未来的临床前景。     

Effect of oxygen tension on chondrocyte extracellular matrix accumulation.

Since cartilage is mainly an avascular tissue, chondrocytes exist in a low-level oxygen environment in vivo. In the present study, we investigated the effect of oxygen tension (20%, 5% and 1% gas phase oxygen concentrations) over a 20-day period on the extracellular matrix accumulation of bovine articular chondrocytes in confluent surface cultures. Matrix accumulation was assessed by the amount of glycosaminoglycan and collagen deposited in the matrix. From initially confluent monolayers, the chondrocytes became distributed throughout a thick layer of extracellular matrix, thus forming a multicell-layer of tissue. Cells maintained their normal rounded shape, indicative of the differentiated phenotype, throughout the 20-day culture period. On a per culture and a per cell basis, the amount of collagen and glycosaminoglycan accumulation in the matrix was lower at the reduced oxygen tensions. Specifically, in 1% oxygen, matrix GAG content reached a steady-state level, with no net increase in GAG levels after two weeks, whereas in 20% oxygen, matrix GAG increased with time. It is concluded that oxygen has a significant effect on the amount of macromolecules accumulated in the extracellular matrix. The implications of these findings in growing cartilage constructs in vitro are discussed.     

Influence of hydrogel mechanical properties and mesh size on vocal fold fibroblast extracellular matrix production and phenotype

Current clinical management of vocal fold (VF) scarring produces inconsistent and often suboptimal results. Researchers are investigating a number of alternative treatments for VF lamina propria (LP) scarring, including designer implant materials for functional LP regeneration. In the present study, we investigate the effects of the initial scaffold elastic modulus and mesh size on encapsulated VF fibroblast (VFF) extracellular matrix (ECM) production toward rational scaffold design. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were selected for this study since their material properties, including mechanical properties, mesh size, degradation rate and bioactivity, can be tightly controlled and systematically modified. Porcine VFF were encapsulated in four PEGDA hydrogels with degradation half lives of approximately 25 days, but with initial elastic compressive moduli and mesh sizes ranging from approximately 30 to 100kPa and from approximately 9 to 27nm, respectively. After 30 days of static culture, VFF ECM production and phenotype in each formulation was assessed biochemically and histologically. Sulfated glycosaminoglycan synthesis increased in similar degree with both increasing initial modulus and decreasing initial mesh size. In contrast, elastin production decreased with increasing initial modulus but increased with decreasing initial mesh size. Both collagen deposition and the induction of a myofibroblastic phenotype depended strongly on initial mesh size but appeared largely unaffected by variations in initial modulus. The present results indicate that scaffold mesh size warrants further investigation as a critical regulator of VFF ECM synthesis. Furthermore, this study validates a systematic and controlled approach for analyzing VFF response to scaffold properties, which should aid in rational scaffold selection/design.     

Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture

Fibrillar collagen gels, which are used extensively in vitro to study tumor-microenvironment interactions, are composed of a cell-instructive network of interconnected fibers and pores whose organization is sensitive to polymerization conditions such as bulk concentration, pH, and temperature. Using confocal reflectance microscopy and image autocorrelation analysis to quantitatively assess gel microarchitecture, we show that additional polymerization parameters including culture media formulation and gel thickness significantly affect the dimensions and organization of fibers and pores in collagen gels. These findings enabled the development of a three-dimensional culture system in which cell-scale gel microarchitecture was decoupled from bulk gel collagen concentration. Interestingly, morphology and migration characteristics of embedded MDA-MB-231 cells were sensitive to gel microarchitecture independently of collagen gel concentration. Cells adopted a polarized, motile phenotype in gels with larger fibers and pores and a rounded or stellate, less motile phenotype in gels with small fibers and pores regardless of bulk gel density. Conversely, cell proliferation was sensitive to gel concentration but not microarchitecture. These results indicate that cell-scale gel microarchitecture may trump bulk-scale gel density in controlling specific cell behaviors, underscoring the biophysical role of gel microarchitecture in influencing cell behavior.     

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.     

细胞外基质因子在兔软骨细胞损伤模型中的表达变化

目的:研究细胞外基质因子在兔关节软骨细胞损伤后不同时间点的表达变化水平。方法:通过机械划伤制备关节软骨细胞损伤模型,观察软骨细胞在划伤后的形态变化。采用实时荧光定量PCR检测软骨细胞在划伤前与划伤后1、3、5 d和7 d共5个时间点的基质金属蛋白酶13(MMP13)及基质金属蛋白酶抑制物1(TIMP1)、透明质酸和Ⅱ型胶原(ColⅡ)基因mRNA表达水平。结果:成功建立了兔关节软骨细胞损伤模型。与划伤前相比,MMP13基因水平在细胞损伤第1天表达明显下降,随后出现升高趋势,第5天达到最高后逐渐下降。TIMP1基因表达量损伤后先上升至第1天然后下降,在第3天降至最低,随后呈现升高趋势。透明质酸和Col-Ⅱ基因水平第1天表达明显降低,随后呈上升趋势,透明质酸mRNA表达量至第5天达到最高,然后逐渐下降。结论:MMP13、TIMP1、透明质酸和Col-Ⅱ基因在细胞损伤后的表达规律相异,为软骨细胞外基质因子表达与软骨细胞之间关系提供实验依据。     

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, (1)H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5-1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.     

Modulation of leukocyte behavior by an inflamed extracellular matrix

Inflammation is a response of the immune system to foreign insult or physical damage. Various cellular and humoral components of the immune system are recruited from the vascular system and are translocated through endothelium, and into extracellular matrix (ECM) compartments of inflamed tissues. This translocation is orchestrated by various types of accessory signals, in the form of soluble or complexed molecules, which evoke remarkable transitions in leukocyte activities. Recruited inflammatory cells give rise to mechanisms of migration, including the secretion of enzymes and other pro-inflammatory mediators and the alteration of their adhesive contacts with the ECM. Hence, migrating cells secrete enzymes, chemokines, and cytokines which interact with the ECM, and thereby, provide the cells with intrinsic signals for coordinating their responses. Resultant products of enzymatic modifications to the ECM microenvironment, such as cytokine- and ECM-derived molecules, may be also part of a cell-signaling mechanism that provides leukocytes with information about the nature of their inflammatory activity; such a mechanism may give the immune system data that can be cognitively interpreted for consequential activities. This article reviews the findings that support this notion and describe the dynamic interactions between participants of the inflammatory processes.     

Substrate chemistry influences the morphology and biological function of adsorbed extracellular matrix assemblies

In addition to mediating cell signalling events, native extracellular matrix (ECM) assemblies interact with other ECM components, act as reservoirs for soluble signalling molecules and perform structural roles. The potential of native ECM assemblies in the manufacture of biomimetic materials has not been fully exploited due, in part, to the effects of substrate interactions on their morphology. We have previously demonstrated that the ECM components, fibrillin and type VI collagen microfibrils, exhibit substrate dependent morphologies on chemically and topographically variable heterogeneous surfaces. Using both cleaning and coating approaches on silicon wafers and glass coverslips we have produced chemically homogeneous, topographically similar substrates which cover a large amphiphilic range. Extremes of substrate amphiphilicity induced morphological changes in periodicity, curvature and lateral spreading which may mask binding sites or disrupt domain structure. Biological functionality, as assayed by the ability to support cell spreading, was significantly reduced for fibrillin microfibrils adsorbed on highly hydrophilic substrates (contact angle 20.7 degrees) compared with less hydrophilic (contact angle 38.3 degrees) and hydrophobic (contact angle 92.8 degrees) substrates. With an appropriate choice of surface chemistry, multifunctional ECM assemblies retain their native morphology and biological functionality.     

Effects of extracellular matrix analogues on primary human fibroblast behavior

In vitro cell culture is a vital research tool for cell biology, pharmacology, toxicology, protein production, systems biology and drug discovery. Traditional culturing methods on plastic surfaces do not accurately represent the in vivo environment, and a paradigm shift from two-dimensional to three-dimensional (3-D) experimental techniques is underway. To enable this change, a variety of natural, synthetic and semi-synthetic extracellular matrix (ECM) equivalents have been developed to provide an appropriate cellular microenvironment. We describe herein an investigation of the properties of four commercially available ECM equivalents on the growth and proliferation of primary human tracheal scar fibroblast behavior, both in 3-D and pseudo-3-D conditions. We also compare subcutaneous tissue growth of 3-D encapsulated fibroblasts in vivo in two of these materials, Matrigel and Extracel. The latter shows increased cell proliferation and remodeling of the ECM equivalent. The results provide researchers with a rational basis for selection of a given ECM equivalent based on its biological performance in vitro and in vivo, as well as the practicality of the experimental protocols. Biomaterials that use a customizable glycosaminoglycan-based hydrogel appear to offer the most convenient and flexible system for conducting in vitro research that accurately translates to in vivo physiology needed for tissue engineering.     

Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation

Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber ("static silicone" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes ("continuous expansion" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype.     

Bovine megakaryocyte integrins: their association with extracellular matrix in vivo

In an effort to define the megakaryocyte microenvironment and the megakaryocyte integrin receptors which might interact with this environment, we undertook a detailed immunofluorescence and immunogold study of bovine bone marrow. Examination of bovine bone marrow using antibodies to laminin, fibronectin and type IV collagen revealed a highly specialised microenvironment with all matrix proteins being present at the basement membranes of fat-cells, vascular sinuses and blood vessels, as well as at the interface with megakaryocytes. In addition, elements of the marrow stroma were heavily labelled by antibody to fibronectin. Lighter labelling was also observed with antibodies to type IV collagen. Immunofluorescence studies were conducted using antibodies to the late antigen (VLA) subgroup of the integrin super family which are receptors for mature proteins. Specifically, antibodies to the a antibodies to the subunit of VLA-2 (collagen), VLA-5 (fibronectin) and VLA-6 (laminin) demonstrated that all of these integrin subunits were diffusely present throughout the megakaryocyte. Antibody staining with the common ₁ subunit for these integrins revealed intense staining of megakaryocyte cytoplasm. Confocal examination of ₁ stained marrow demonstrated a clear punctate distribution with equal intensity from the perinuclear zone through to the peripheral zone. These data, as well as in vitro data generated from our laboratory and others, suggest that the specialised megakaryocyte microenvironment and its interaction with the cell's integrins may localise megakaryocytes to the abluminal side of the vascular sinus, thus positioning them for further interaction with the sinus.     

A thermo-sensitive poly(organophosphazene) hydrogel used as an extracellular matrix for artificial pancreas

A poly(organophosphazene) bearing alpha-amino-omega-methyl-poly(ethylene glycol) (AMPEG) and hydrophobic L-isoleucine ethyl ester (IleOEt) side groups has been synthesized. This material exhibited 4 phase transitions in an aqueous solution on gradually increasing the temperature, i.e., a transparent sol, a transparent gel, an opaque gel and a turbid sol. A 10 wt% buffered solution of the polymer was employed to entrap islets of Langerhans in an artificial pancreas. Rat islets entrapped in the gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free rat islets and islets entrapped in other types of polymer gels. Over a 28-day culture period, the rat islets in the poly(organophosphazene) hydrogel maintained higher cell viability and insulin production versus rat islets in different hydrogels and free islets. This thermo-sensitive injectable, biodegradable matrix can be used with several cell types, including nerve cells, to promote nerve regeneration.     

Effects of immunosuppressors and cytostatics on the extracellular matrix production in experimental nephropathy

Methylprednisolone increased the content of laminine, cyclosporin increased the content of fibronectin, and cyclophosphamide suppressed the extracellular matrix production in experimental nephrotoxic nephritis and puromycin aminonucleoside nephrosis. Therapy should be chosen with due consideration of the extracellular matrix structure in different morphological variants of chronic glomerulonephritis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 11, pp. 584–587, November, 1998     

Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells

Biomaterials developed for tissue engineering and wound healing applications need to support robust cell adhesion, yet also need to be replaced by new tissue synthesized by those cells. In order to maintain mechanical integrity of the tissue, the cells must generate sufficient extracellular matrix before the scaffold is degraded. We have previously shown that materials containing cell adhesive ligands to promote or improve cell adhesion can decrease extracellular matrix production (Mann et al., Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999;20:2281-6). Such decreased matrix production by cells in tissue engineering scaffolds may result in tissue failure. However, we have found that TGF-beta1 can be used in scaffolds to dramatically increase matrix production. Matrix production by vascular smooth muscle cells grown on adhesive ligand-modified glass surfaces and in PEG hydrogels containing covalently bound adhesive ligands was increased in the presence of 0.04 pmol/ml (1 ng/ml) TGF-beta1. TGF-beta1 can counteract the effect of these adhesive ligands on matrix production; matrix production could be increased even above that observed in the absence of adhesive peptides. Further, TGF-beta1 covalently immobilized to PEG retained its ability to increase matrix production. Tethering TGF-beta1 to the polymer scaffold resulted in a significant increase in matrix production over the same amount of soluble TGF-beta1.     

Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.