Engineered cartilage via self-assembled hMSC sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-β1

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Abstract

Self-assembling cell sheets have shown great potential for use in cartilage tissue engineering applications, as they provide an advantageous environment for the chondrogenic induction of human mesenchymal stem cells (hMSCs). We have engineered a system of self-assembled, microsphere-incorporated hMSC sheets capable of forming cartilage in the presence of exogenous transforming growth factor β1 (TGF-β1) or with TGF-β1 released from incorporated microspheres. Gelatin microspheres with two different degrees of crosslinking were used to enable different cell-mediated microsphere degradation rates. Biochemical assays, histological and immunohistochemical analyses, and biomechanical testing were performed to determine biochemical composition, structure, and equilibrium modulus in unconfined compression after 3 weeks of culture. The inclusion of microspheres with or without loaded TGF-β1 significantly increased sheet thickness and compressive equilibrium modulus, and enabled more uniform matrix deposition by comparison to control sheets without microspheres. Sheets incorporated with fast-degrading microspheres containing TGF-β1 produced significantly more GAG and GAG per DNA than all other groups tested and stained more intensely for type II collagen. These findings demonstrate improved cartilage formation in microsphere-incorporated cell sheets, and describe a tailorable system for the chondrogenic induction of hMSCs without necessitating culture in growth factor-containing medium.

Introduction

Osteoarthritis (OA) is a degenerative disease of the articular cartilage affecting millions of people worldwide [1]. As no current treatment can fully and consistently restore normal joint function to patients afflicted with OA [2], there is a significant clinical need for alternative therapies for cartilage regeneration. Many approaches to the tissue engineering of articular cartilage involve the use of cells in combination with soluble bioactive factors and biomaterials that may provide specific microenvironmental cues for chondrogenic induction [3], [4], [5]. Mesenchymal stem cells (MSCs) from bone marrow have been shown to be a promising cell source for these cartilage tissue engineering strategies, as they can be expanded in culture without losing multipotency, and can differentiate into many cell types of the connective tissue lineage including chondrocytes under appropriate conditions [6]. Specifically, two important factors for the in vitro chondrogenic induction of MSCs are high initial cell density and exposure to transforming growth factor β (TGF-β) [7], [8], [9].

Several in vitro culture methods have been developed for MSC chondrogenesis, including aggregate or pellet culture [9], [10], [11], micromass culture [12], [13], and self-assembling cell sheet systems [7], [8], [14], [15]. These culture systems take advantage of the abundant cell–cell interactions that occur in 3D high density culture, without the potential interference of a biomaterial scaffold. In particular, self-assembling cell sheets show promise for use in cartilage tissue engineering applications, as they may form larger constructs with much greater surface areas and volumes than aggregates or tiny micromass cultures [8], [15]. Unlike spherical cell aggregates, which are limited in size by the diffusion distance of nutrients into the center of the sphere, flat sheets of various dimensions can be formed without necessitating a proportional increase in construct thickness, enabling nutrient diffusion to all regions of the tissue. Upon surgical evaluation, chondral defects in the knee have an area of at least 0.5 cm2, with over a third of the defects having areas of at least 1 cm2 [16]. Self-assembling sheets could be clinically practical for the treatment of these defects, as sheets of the appropriate size could be formed and then implanted into a defect as an intact piece. This is in contrast to smaller cell constructs, which may not be as readily applied for the clinical treatment of cartilage defects since a number of constructs would be required to fill a single lesion. It may be difficult to localize multiple constructs to a defect, and in order to repair the damaged cartilage, the individual cell constructs would have to integrate with each other as well as with the surrounding host tissue.

Though MSC sheets of adequate size can be formed through self-assembly methods, mechanical stability can be a problem in high density cell systems, particularly at early time points in vitro. An ideal engineered cartilage construct would have the strength necessary to withstand mechanical forces in the joint until the regenerated cartilage gains adequate mechanical properties to support the tissue [17]. Additionally, constructs should be sturdy enough to be easily manipulated and implanted without losing their shape [18]. A major advantage of “scaffold-free” cell systems over traditional polymer scaffold-based constructs is the lack of excess amounts of polymer material, which eliminates problems including slow polymer degradation, potential toxicity, and interference with cell–cell contacts [7], [15], [19], [20]. However, “scaffold-free” construct approaches lack some crucial benefits of polymer scaffolds including shape, structure, and mechanical stability. During the first few weeks of culture, before abundant ECM components are produced, densely cellular constructs are typically fragile and exhibit poor mechanical properties [18], [21]. The mechanical properties of these tissues may improve over extended periods of in vitro culture with continuous supplementation of TGF-β [7], but the lengthy culture requirements are expensive and time-intensive, and may be prohibitive to the clinical translation of this technology.

A minimum 3-week culture requirement is typical for high density MSC systems, which need extended periods of growth factor supplementation in order to differentiate and develop a neocartilaginous extracellular matrix (ECM). The ECM growth and maturation occurring during the culture period is beneficial to the mechanical stability of the construct, however, new problems arise as the tissue volume increases. Non-uniform spatial growth factor delivery occurs due to diffusional limitations of TGF-β from the culture media to cells in central regions of the tissue as well as growth factor uptake by cells in the exterior tissue regions. Uneven delivery of growth factor can lead to non-uniform patterns of differentiation throughout the tissue bulk, and in some cases necessitates an increase in growth factor concentration in the culture medium to achieve chondrogenesis in the construct interior [10].

To address many of the problems with current high density MSC systems, we have developed a system of self-assembling MSC sheets incorporated with growth factor releasing hydrogel microspheres. The inclusion of biodegradable gelatin microspheres within MSC sheets could balance the need for quickly-degrading scaffolds of limited mass with the structural advantages of an incorporated biomaterial. Gelatin is uniquely suited for this application because it is a biocompatible, biodegradable hydrogel that facilitates sustained delivery of certain growth factors including TGF-β1 at rates adjustable by controlling the rate of polymer degradation, which in turn can be controlled by the degree of polymer crosslinking [22], [23], [24]. When distributed within self-assembling MSC sheets, TGF-β1 loaded gelatin microspheres could uniformly deliver chondrogenic growth factor directly to the interior regions of the sheets over a sustained period, enabling spatially homogenous differentiation at rates tailorable by adjusting the microsphere crosslinking levels. Additionally, this system could potentially reduce in vitro culture time, as the need for extended periods of exogenous growth factor supplementation would be eliminated.

Here, we describe a system of self-assembled, microsphere-incorporated human MSC (hMSC) sheets capable of forming cartilage in the presence of exogenous TGF-β1 or with TGF-β1 released from the incorporated microspheres. Our hypothesis was that the incorporation of gelatin microspheres with or without growth factor into hMSC sheets could improve both the mechanical properties and spatial distribution of neocartilage matrix. We also hypothesized that TGF-β1 loaded microspheres could enable enhanced chondrogenesis in hMSC sheets without requiring exogenous growth factor supplementation. Gelatin microspheres with two different degrees of genipin crosslinking enabled elucidation of the roles of different microsphere degradation and growth factor release rates on chondrogenesis within the system.

Section snippets

hMSC isolation and culture

Bone marrow aspirates from the posterior iliac crest of healthy donors were obtained under a protocol approved by the University Hospitals of Cleveland Institutional Review Board and processed by the Skeletal Research Center Mesenchymal Stem Cell Core Facility as previously described [25]. Briefly, the aspirates were washed with growth medium, which was comprised of low glucose Dulbecco's modified Eagle's medium (DMEM-LG; Sigma) containing 10% pre-screened fetal bovine serum (FBS) [26].

Microsphere characterization

Hydrated gelatin microspheres were roughly spherical, with smooth surfaces (Fig. 1). The diameters of low and high Gp microspheres did not significantly differ, while the degrees of crosslinking between microsphere groups were significantly different, with the low Gp group 28.3 ± 7.2% crosslinked and the high Gp group 67.6 ± 4.5% crosslinked (Table 1). High Gp microspheres were a dark blue color as a result of the genipin crosslinking reaction [36]. Growth factor release from crosslinked

Discussion

The aim of this study was to engineer self-assembling hMSC constructs containing biodegradable microspheres with or without chondrogenic growth factor, enabling improved neocartilage matrix formation and mechanical properties without requiring exogenous growth factor supplementation. In contrast to our previous work involving hMSC aggregates incorporated with TGF-β1 releasing PLGA microspheres [39], this system of gelatin microsphere-incorporated sheets enables the formation of larger

Conclusions

This study demonstrates the utility of growth factor-incorporated microspheres as a means of enhancing neocartilage tissue formation in high-density hMSC culture. As evaluated via biochemical assays, histological and immunohistochemical analysis, and biomechanical testing, incorporation of growth-factor releasing microspheres into hMSC sheets enhances the structure and function of the high density cell sheets. Beyond producing sheets with superior mechanical properties and more uniform matrix

Acknowledgments

The authors would like to thank Angela Carlson, Amad Awadallah and Adam Whitney for technical assistance. This work was supported by NIH/NIAMS T32 AR007505 (LDS), a National Science Foundation Graduate Research Fellowship (PND), Biomedical Research and Technology Transfer Grant 09-071 from the Ohio Department of Development (EA) and a New Scholar in Aging grant from the Ellison Medical Foundation (EA).

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