Regeneration enhanced in critical-sized bone defects using bone-specific extracellular matrix protein

Abstract

Extracellular matrix (ECM) products have the potential to improve cellular attachment and promote tissue-specific development by mimicking the native cellular niche. In this study, the therapeutic efficacy of an ECM substratum produced by bone marrow stem cells (BM-MSCs) to promote bone regeneration in vitro and in vivo were evaluated. Fluorescence-activated cell sorting analysis and phenotypic expression were employed to characterize the in vitro BM-MSC response to bone marrow specific ECM (BM-ECM). BM-ECM encouraged cell proliferation and stemness maintenance. The efficacy of BM-ECM as an adjuvant in promoting bone regeneration was evaluated in an orthotopic, segmental critical-sized bone defect in the rat femur over 8 weeks. The groups evaluated were either untreated (negative control); packed with calcium phosphate granules or granules+BM-ECM free protein and stabilized by collagenous membrane. Bone regeneration in vivo was analyzed using microcomputed tomography and histology. in vivo results demonstrated improvements in mineralization, osteogenesis, and tissue infiltration (114 ± 15% increase) in the BM-ECM complex group from 4 to 8 weeks compared to mineral granules only (45 ± 21% increase). Histological observations suggested direct apposition of early bone after 4 weeks and mineral consolidation after 8 weeks implantation for the group supplemented with BM-ECM. Significant osteoid formation and greater functional bone formation (polar moment of inertia was 71 ± 0.2 mm⁴ with BM-ECM supplementation compared to 48 ± 0.2 mm⁴ in untreated defects) validated in vivo indicated support of osteoconductivity and increased defect site cellularity. In conclusion, these results suggest that BM-ECM free protein is potentially a therapeutic supplement for stemness maintenance and sustaining osteogenesis.

FIGURE 1

The total cell versus absolute SSEA-4 number after 14 days of cell culture on tissue culture plastic (TCP) and different BM-ECM concentrations (10, 20, 40 μg/mL). The percentage indicates the ratio of SSEA-4 (%) in total cell number. SSEA-4 cell growth was used as a marker of cells’ stemness. Results show a significantly different (*p < 0.05) cell growth of SSEA-4 and total cell number between TCP and other groups. BM-ECM, bone marrow specific extra cellular matrix; SSEA-4, stage-specific embryonic antigen-4

FIGURE 2

Radiographic observation on control, granules, and granules+ BM-ECM groups after 2, 4, 6, and 8 weeks. Images were captured with a scanner at 100 kV source voltage and 100 mA source current with a 0.05 mm aluminum filter and a spatial resolution of 8.77 μm. Healing process is noted during 8 weeks according to the callus tissue reflections in images. Collagen wrap was secured during 8 weeks and the presence of granules was observed in between the defect site in groups with granules and granules with BM-ECM. BM-ECM, bone marrow specific extra cellular matrix

FIGURE 3

Cross section areas from the μCT images (a) for empty defect, with granules, and granules with BM-ECM groups after 4 and 8 weeks. High contrast areas show the mineralized areas. 3D reconstructions from μCT results (b) from the μCT images for empty defect, with granules, and granules with BM-ECM groups after 4 and 8 weeks. Collagen wraps are pseudo-colored purple, mineralized tissue and granules in the defect site were colored in blue wires are colored yellow. μCT, microcomputed tomography; BM-ECM, bone marrow specific extra cellular Matrix

FIGURE 4

Bone quantity and bone quality calculated for empty defects, granules, and granules with BM-ECM after 4 and 8 weeks. (a) Bone quantity is reported as the regenerated bone volume (mm³) by showing osteoid and dense volumes. (b) Bone quality is reported as the bone mineral density (Gy). BMD was significantly different (p < 0.05) from 4 to 8 weeks in all groups. Bone volume regeneration was no significantly different within the groups.BM-ECM, bone marrow specific extra cellular matrix

FIGURE 5

Effect of BM-ECM on bone increase from 4 to 8 weeks in samples with granules and granules with BM-ECM. (a) Bone increase percentage (%) from 4 to 8 weeks between only granules and granules with BM-ECM. The results show a significant increase in bone percentage between the groups (p < 0.05). (b) Functionality of metrics based on polar moment of inertia (mm⁴) in granules and granules with BM-ECM groups after 4 and 8 weeks. The results in both groups are significantly different within the groups from week 4 to 8 (p < 0.01). BM-ECM, bone marrow specific extra cellular matrix

FIGURE 6

Histological evaluation for empty defects, granules, and granules with BM-ECM after 4 and 8 weeks. The longitudinal sections were prepared and stained by Paragon and Analine Blue to label calcium, connective tissue, and collagen type I. The images show the bone regeneration, mineralized collagen wrap, and connective tissue formation in different magnification ×4 and ×10. Empty defect area shows a clear center with minimized calcification around the defect sites. Defect sites with granules and granules with BM-ECM show more calcification and the presence of more connective tissue after 8 weeks. Bridging is noticeable in groups with granules and BM-ECM. The scale bar is equal to 1 cm. BM-ECM, bone marrow specific extra cellular matrix