Elsevier

Biomaterials

Volume 24, Issue 15, July 2003, Pages 2593-2603
Biomaterials

Comparison of human bone marrow stromal cells seeded on calcium-deficient hydroxyapatite, β-tricalcium phosphate and demineralized bone matrix

https://doi.org/10.1016/S0142-9612(03)00062-0Get rights and content

Abstract

The aim of this study was to compare three resorbable biomaterials regarding seeding efficacy with human bone marrow stromal cells (BMSCs), cell penetration into the matrix, cell proliferation and osteogenic differentiation. Calcium-deficient hydroxyapatite (CDHA), β-tricalcium phosphate (β-TCP), and demineralized bone matrix (DBM) were seeded with human BMSCs and kept in human serum and osteogenic supplements for 3 weeks. Morphologic and biochemical evaluations were performed on day 1, 7, 14 and 21. The allograft DBM and CDHA exhibited both an excellent seeding efficacy while the performance of β-TCP was lower when compared. The total protein content and the values for specific alkaline phosphatase (ALP) increased on all matrices and no significant difference was found for these two markers. BMSCs in monolayer had a significant increase of protein, but not of ALP. Osteocalcin (OC) values increased significantly higher for BMSC in cultures on DBM when compared to CDHA and β-TCP. The OC levels decreased significantly in the BMSC monolayer culture. BMSCs were found inconsistently within the synthetic materials, whereas in DBM they were found more homogeneously distributed throughout the matrix. All three matrices promoted BMSC proliferation and differentiation to osteogenic cells. DBM allografts seem to be more favorable with respect to cell ingrowth tested by histology, and osteogenic differentiation ascertained by an increase of OC. CDHA with its high specific surface area showed more favorable properties than β-TCP regarding reproducibility of the seeding efficacy.

Introduction

Repair of large bone defects represents a challenge to orthopedic surgeons since autogenous grafts are not available in large amounts and its removal causes morbidity at the donor site [1], [2], [3]. Numerous animal studies demonstrated that seeding periosteal cells or bone marrow stromal cells (BMSCs) on suitable matrices prior to implantation can improve healing of large bone defects [4], [5], [6]. A new tissue-engineering approach is to cultivate BMSCs on matrices to further increase the bioactivity of the cell-matrix composite before implantation [7], [8], [9], [10], [11], [12], [13], [14].

Ideally, the matrix serves as a scaffold that is subsequently replaced by newly formed bone in vivo. Therefore, resorbable materials are favored that provide a certain 3D stability [15], [16], [17].

Both, synthetic and allograft materials, are available at present. From the group of the synthetic materials, many ceramics have proven to be biocompatible. Key factors seem to be their ability to bond bone minerals directly and to promote new bone formation by osteoconduction [18], [19]. Among the resorbable ceramics are materials such as β-tricalcium phosphate (β-TCP), and a recently emerging group of ceramics called low-temperature apatites (LTA) (Fig. 1A). These LTA are produced from reactive calcium phosphates at room temperature. In contrast to most apatite compounds, these LTA are not sintered. As a result, LTA possess a very large specific surface area (20–100 m2/g), similar to that observed in bone apatite crystals (Fig. 1B). The large specific surface area makes these apatites crystals resorbable [20].

So far, most in vivo studies on LTA have been devoted to calcium phosphate cements, which do not have an open-macroporous structure. Here, open-macroporous LTA were obtained via the so-called calcium phosphate emulsions [21].

Ceramics possess osteoconductive properties, but they do not have intrinsic osteoinductive capacity [8]: they are unable to induce new bone formation in extraosseous sites. This incapacity of inducing bone formation on its own can be overcome by seeding osteoprogenitor cells onto ceramic matrices [7], [8], [11], [13], [14].

Demineralized bone matrix (DBM)—as an allogenous material—contains bone morphogenic and matrix proteins in contrast to ceramic materials. Bone morphogenic proteins (BMP) are potent osteoinductive glycoproteins while matrix proteins, such as different collagens provide an osteoconductive matrix [4]. DBM seeded with bone marrow cells seems to accelerate the healing of critical size bone defects in rats [4]. It is unknown whether in vitro cultivation with BMSCs prior to implantation is feasible or whether it provides advantages for healing bone defects.

The aim of the current study was to evaluate the properties of three different resorbable biomaterials with regard to seeding efficacy, cell penetration into the matrices, cell proliferation, and osteogenic differentiation. Two synthetic biomaterials, β-TCP and a new biomaterial called CDHA (both by the Dr. h.c. Robert Mathys Foundation) and the allograft material DBM (Grafton® Flex by Osteotech) were seeded with human BMSCs and kept in an in vitro culture for up to 3 weeks.

Section snippets

Materials

The β-tricalcium phosphate (β-TCP) and calcium-deficient hydroxyapatite (CDHA), Ca9(PO4)5(HPO4)OH, ceramic bodies (both Robert Mathys Foundation, Switzerland) were produced in an emulsion process as described earlier [21]. β-TCP has an overall porosity of 84 vol% with an average macroporosity (pores ∅0.2–0.6 mm) of 55 vol% and a microporosity (pores ∅<5 μm) of 29 vol%. The specific surface area is 0.5 m2/g. In contrast, CDHA has a total porosity of 85 vol%, 54% for macropores (∅0.2–0.6 mm) and 31% for

Seeding efficacy

The three matrices exhibited significant differences in seeding efficacy (Table 1). 4.5% of the total number of cells did not adhere to CDHA. In contrast, β-TCP samples displayed a rather low seeding efficacy with 41.5% cells not adhering to the biomaterial. However, it should be noted that the standard deviation was also high, about 33%. DBM proved to be an excellent matrix for BMSCs. All cells adhered to the matrix after seeding at day 1. There was a significant difference of seeding efficacy

Discussion

At present, numerous types of biomaterials are available on the market. They can be divided into resorbable and non- or minimally resorbable biomaterials [19]. Resorbable materials can further be divided into osteoconductive and osteoinductive regarding their ability to allow or induce bone formation, respectively [24], [25], [26].

There are numerous advantages and disadvantages of the different scaffolds: e.g. scaffolds made of polymers derived from lactide and/or glycolide (PLA/PGA) for

Conclusion

All tested biomaterials had a significant increase of protein and osteogenic differentiation markers in the experimental setting. However, the increase of protein or specific ALP did not significantly differ between the groups in a static in vitro cultivation period of 21 days. Osteocalcin on day 21 was significantly higher on DBM than on the ceramic matrices. Comparing the synthetic materials, CDHA appeared to have more favorable properties than β-TCP thanks to a more reproducible seeding

Acknowledgements

We would like to thank Ms. Iris Albers and Ms. Nicole Brauer-Dewor for their excellent work in the laboratory during cell culturing and obtaining the results. We also would like to thank Sven Schneider, PhD, for his excellent statistic analysis.

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    The study was financially supported by a research grant of the Dr. h.c. Robert Mathys foundation.

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