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. 2015 Nov;26(11):254.
doi: 10.1007/s10856-015-5591-3. Epub 2015 Oct 8.

Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids

Hideyuki Suenaga  1 , Katsuko S Furukawa  2 , Yukako Suzuki  3 , Tsuyoshi Takato  3   4 , Takashi Ushida  5
Affiliations

Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids

Hideyuki Suenaga et al. J Mater Sci Mater Med. 2015 Nov.

Abstract

Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, β-tricalcium phosphate (β-TCP) granules, or β-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60% those of native bones, respectively). All tests showed poor bone regeneration in the β-TCP and β-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

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Figures

Fig. 1
Fig. 1
Outline of the experimental design: human mesenchymal stem cells (MSCs) were obtained, cultured in vitro for 10 days, and frozen in aliquots until further use. When required, the frozen stock was thawed and cultured for 7 days, followed by rotation culture for 1 day. The resulting MSC spheroids were collected and implanted into calvarial bone defects created in rats (n = 10). Three treatment groups with the following implants were analyzed for bone regeneration: MSC spheroids, beta-tricalcium phosphate (β-TCP), and a combination of MSC spheroids + β-TCP (n = 10 rats per treatment group). After a recovery period of 8 weeks, bone regeneration at the defect sites was evaluated
Fig. 2
Fig. 2
Implantation of mesenchymal stem cell (MSC) spheroids in rat calvarial defects: calvarial defects were generated bilaterally in each rat, and MSC spheroids were implanted on the left side, whereas the right side was left untreated as control
Fig. 3
Fig. 3
Characteristics of the mesenchymal stem cell (MSC) spheroids: a light microcopy image showing free-floating multicellular spheroids formed from human bone marrow-derived mesenchymal stem cells in rotation culture (1 day), without the use of any scaffold. b Phase-contrast microscopy of spheroids obtained from human MSCs after 1 day in rotation culture (scale, 100 μm). c Fluorescence microscopy of spheroids stained with calcein-AM to visualize live cells that appear green. d Fluorescence microscopy of spheroids stained with propidium iodide to visualize the nucleus (red) of dead cells (Color figure online)
Fig. 4
Fig. 4
Histological assessment of bone regeneration at 8 weeks post-implantation: Bone regeneration was examined in calvarial bone defects at 8 weeks after implantation of mesenchymal stem cell (MSC) spheroids, β-TCP granules alone, and MSC spheroids + β-TCP, and was compared with the untreated defect sites. a Hematoxylin and eosin (HE) staining of the whole bone section showing the implanted area (left side) and the untreated control area (right side). b, c Magnified view of the HE-stained, MSC spheroid-implanted site (b) and control site (c). The control site showed only a thin band of fibrous connective tissue in the defect area along with minimal new bone formation (c). In contrast, at the MSC spheroid-implanted site, new vascularization was apparent, along with a significant amount of new bone and bone proteins throughout the defect area (b). d, e Immunohistochemical staining to visualize distribution of osteocalcin, MSC-implanted site (d) and untreated defect site (e). f, g Immunohistochemical staining to visualize distribution of osteopontin in the MSC-implanted site (f) and untreated defect site (g). h, i Defect site implanted with β-TCP granules alone (h) and β-TCP + MSC spheroids (i). The β-TCP implant site showed disintegrating tissue, fibrous tissue, and blood vessels between β-TCP granules (shown by asterisks). The site implanted with spheroids + β-TCP showed formation of new bone with fewer interspersed β-TCP granules (shown by asterisks). Scale, 500 μm
Fig. 5
Fig. 5
Micro-computed tomography (CT) images of a rat skull at 8 weeks after implantation: Calvarial defects created bilaterally in rats were implanted with mesenchymal stem cell (MSCs) spheroids (left side) or were left untreated (right side). At 8 weeks post-implantation, the defect sites were examined by micro-CT. At the implant site, the diameter of the cylindrical holes had narrowed, (left side), whereas at the control site, there was significantly less bone regeneration (right side) (a). Micro-computed tomography (CT) images of defect site implanted with β-TCP granules alone (b), micro-computed tomography (CT) images of defect site implanted with β-TCP granules and MSC spheroids (c)
Fig. 6
Fig. 6
Bone tissue spectra obtained by Raman spectroscopy: a Raman spectrum of newly formed bone from a mesenchymal stem cell (MSC) spheroid-implanted calvarial defect site. In these spectra, the background signals have been removed. b A typical Raman spectrum of a native calvarial bone showing the major peaks
Fig. 7
Fig. 7
Measurement of dynamic mechanical properties of newly formed bone: Mechanical performance of bone samples obtained from the implantation sites of MSC spheroids, β-TCP, and MSC spheroids + β-TCP was compared with that of the native bone at 8 weeks post-implantation. a Maximum bending stress of the indicated bone samples. Values plotted are mean ± standard error (SE) (n = 5 in each group). *P < 0.05,**P < 0.01, ***P < 0.005 versus native bone; # P < 0.05, ## P < 0.01 versus MSC spheroid group. b Young’s modulus for the indicated bone samples. Values plotted are mean ± SE (n = 5 in each group). *P < 0.05, **P < 0.01, ***P < 0.005 versus native bone; # P < 0.05, ## P < 0.01 versus MSC spheroid group
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