Elsevier

Biomaterials

Volume 26, Issue 25, September 2005, Pages 5158-5166
Biomaterials

Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold

https://doi.org/10.1016/j.biomaterials.2005.01.002Get rights and content

Abstract

Functional engineering of musculoskeletal tissues generally involves the use of differentiated or progenitor cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. We have previously shown that electrospun, three-dimensional nanofibrous scaffolds that morphologically resemble collagen fibrils are capable of promoting favorable biological responses from seeded cells, indicative of their potential application for tissue engineering. In this study, we tested a three-dimensional nanofibrous scaffold fabricated from poly(ε-caprolactone) (PCL) for its ability to support and maintain multilineage differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in vitro. hMSCs were seeded onto pre-fabricated nanofibrous scaffolds, and were induced to differentiate along adipogenic, chondrogenic, or osteogenic lineages by culturing in specific differentiation media. Histological and scanning electron microscopy observations, gene expression analysis, and immunohistochemical detection of lineage-specific marker molecules confirmed the formation of three-dimensional constructs containing cells differentiated into the specified cell types. These results suggest that the PCL-based nanofibrous scaffold is a promising candidate scaffold for cell-based, multiphasic tissue engineering.

Introduction

Tissue engineering approaches are currently being developed to successfully repair and restore the function of damaged or diseased tissues [1], [2], [3]. The basic principle involves the use of an appropriate cell source and a biocompatible and biodegradable scaffold to produce a construct that structurally and functionally mimic the target tissue. An additional challenge is that most tissues and organs are multiphasic in nature and contain multiple cell types. Thus, an ideal biomaterial scaffold should be capable of supporting multilineage cell types. To date, few attempts have been made to engineer tissues consisting of multiple cell types. A successful example for engineered multiphasic tissue is the osteochondral construct that is composed of bone and cartilage tissues [4], [5], [6], [7], [8]. The common approach has been to integrate the chondral construct and the osteo construct after they are separately fabricated from stem cells [6] or differentiated cells [7].

Adult human bone marrow, as well as muscle [9], [10], [11], adipose [12], trabecular bone [13], and a host of other tissues, contain a population of mesenchymal ‘stem-like’ cells with the ability to differentiate into a variety of connective tissue lineages, including adipose, bone, cartilage, muscle, and tendon, when provided with the appropriate culture environment and inductive agents [14], [15]. Additionally, their potential for expansion, and lineage-specific differentiation, combined with derivation from autologous sources, suggest human mesenchymal stem cells (hMSCs) may be candidate cells for tissue engineering and regenerative therapies. The lineage-specific selective differentiation of MSCs has been well demonstrated and documented in vitro. For example, when cultured in the presence of inductive media, containing differentiation-promoting agents and growth factors, hMSCs differentiate into osteoblasts [16], adipocytes [15], and chondrocytes (in the form of high-density cell pellets) [17]. While three-dimensional biomaterial scaffolds have been used to engineer cell-type specific constructs, to our knowledge, there is no report on the successful application of a single three-dimensional scaffold to support cell differentiation along these distinct lineages.

The electrospinning process was originally developed to produce ultra-fine polymer fibers [18], and has recently been used as a novel technique to synthesize scaffolds that mimic the architecture and mechanical properties of the extracellular matrix (ECM) of native tissues. We have previously shown that electrospun three-dimensional nanofibrous structures share morphological similarities to collagen fibrils, and are capable of promoting favorable biological responses from seeded cells. For example, fetal bovine chondrocytes, seeded within nanofibrous scaffolds, maintain their differentiation state through long-term in vitro culture, suggesting the potential application of such scaffolds for tissue-engineering [19], [20].

Ideally, the engineering of functional tissue substitutes would involve the use of a single three-dimensional biocompatible and biodegradable scaffold capable of supporting the selective, lineage-specific differentiation of a single autologous cell source, such as hMSCs. In this study, a three-dimensional nanofibrous scaffold fabricated by electrospinning of a synthetic biodegradable polymer, poly(ε-caprolactone) (PCL) was tested for its ability to support and maintain multilineage differentiation of bone marrow-derived hMSCs in vitro.

Section snippets

Reagents

All reagents were purchased from Sigma Chemicals (St. Louis, MO) unless specified otherwise.

Isolation and culture of bone marrow-derived hMSCs

With approval from the Institutional Review Board of Thomas Jefferson University, bone marrow-derived hMSCs were obtained from the femoral heads of patients undergoing total hip arthroplasty, and processed as previously described [13], [16], [21]. Briefly, whole bone marrow was curetted from the exposed cutting plane of the femoral neck, washed extensively in Dulbecco's Modified Eagle's medium (DMEM;

PCL-based nanofibrous scaffold

Electrospinning of PCL-based nanofibers resulted in a scaffold composed of uniform, randomly oriented fibers of an average diameter of 700 nm, as seen by scanning electron microscopy (SEM; Fig. 1). Following an 8 week incubation in culture medium at 37 °C, scaffolds maintained their integrity and three-dimensional structure, while exhibiting no noticeable change in dry weight over the entire culture period (data not shown).

Morphological analysis

Cellular constructs maintained with or without adipogenic, chondrogenic,

Discussion

A current challenge in tissue engineering is to develop methods for multiphasic tissue constructs. The results reported here are thus of relevance as they clearly demonstrate that adult hMSCs obtained from a single patient and cultured on a biocompatible and biodegradable PCL-based nanofibrous scaffold can be used to engineer adipose, cartilage, and bone, in vitro, when provided with the appropriate inductive agents.

The technique of electrospinning polymers was developed in 1934 by Formhals [18]

Conclusion

The findings reported here indicate that multilineage differentiation of MSCs is fully supported within the nanofibrous scaffold. To our knowledge, this study is the first to report the selective differentiation of hMSCs isolated from a single patient along the adipogenic, chondrogenic, and osteogenic lineages utilizing the same single biocompatible and mechanically sound polymeric matrix. This capability supports the feasibility of tissue-engineering multiphasic constructs using a single cell

Acknowledgements

The authors are grateful to Drs. David Hall, Peter Alexander, and Assia Derfoul for their invaluable assistance and helpful discussion. This work is supported by NIH Z01 AR 41113.

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