Real-time-guided bone regeneration around standardized critical size calvarial defects using bone marrow-derived mesenchymal stem cells and collagen membrane with and without using tricalcium phosphate: an in vivo micro-computed tomographic and histologic experiment in rats

Abstract

The aim of the present real time in vivo micro-computed tomography (µCT) and histologic experiment was to assess the efficacy of guided bone regeneration (GBR) around standardized calvarial critical size defects (CSD) using bone marrow-derived mesenchymal stem cells (BMSCs), and collagen membrane (CM) with and without tricalcium phosphate (TCP) graft material. In the calvaria of nine female Sprague-Dawley rats, full-thickness CSD (diameter 4.6 mm) were created under general anesthesia. Treatment-wise, rats were divided into three groups. In group 1, CSD was covered with a resorbable CM; in group 2, BMSCs were filled in CSD and covered with CM; and in group 3, TCP soaked in BMSCs was placed in CSD and covered with CM. All defects were closed using resorbable sutures. Bone volume and bone mineral density of newly formed bone (NFB) and remaining TCP particles and rate of new bone formation was determined at baseline, 2, 4, 6, and 10 weeks using in vivo µCT. At the 10th week, the rats were killed and calvarial segments were assessed histologically. The results showed that the hardness of NFB was similar to that of the native bone in groups 1 and 2 as compared to the NFB in group 3. Likewise, values for the modulus of elasticity were also significantly higher in group 3 compared to groups 1 and 2. This suggests that TCP when used in combination with BMSCs and without CM was unable to form bone of significant strength that could possibly provide mechanical "lock" between the natural bone and NFB. The use of BMSCs as adjuncts to conventional GBR initiated new bone formation as early as 2 weeks of treatment compared to when GBR is attempted without adjunct BMSC therapy.

Figure 1

Surgical protocol. (a) Reflection of a 15-mm-long midline cutaneous straight incision along the sagital suture over the parietal bone of the scalp and the skin and underlining tissues including the musculature and the periosteum were reflected bilaterally to expose the calvarium. (b) Using a trephine drill, a full-thickness CSD was created on the parietal region lateral to the sagital suture. (c) Full-thickness bone (including the outer and inner cortices) was carefully removed to prevent damage to the dura. (d) A resorbable CM was placed over the defect. (e) Flaps were sutured using resorbable sutures. CM, collagen membrane; CSD, critical size calvarial defect.

Figure 2

In vivo μCT images of rat calvarial models with critical size defects scanned at 2-, 4-, 6-, and 10-week intervals. (a) Control group shows traces of new bone formation over the study period. (b) Formation of new bone (in yellow) at 2, 4, 6, and 10 weeks in critical size defects filled with MSCs and covered with a resorbable CM. (c) Formation of new bone (in yellow) at 2, 4, 6, and 10 weeks in critical size defects filled with β-TCP soaked in MSCs and covered with a resorbable CM. CM, collagen membrane; MSC, mesenchymal stem cell; TCP, tricalcium phosphate.

Figure 3

Histologic assessment (using hematoxylin-eosin staining) of CSD and native calvaria in group 1. (a) Edges of the CSD (red arrows) bridged by fibrous CT with remnants of CM (×4). (b) NFB at the edge of the CSD with fibrous CT at the advancing edge of NFB. Blue arrows show the boundary between NFB and NB with remnants of CM (×10). (c) Osteocytes (green arrows) and BVs are evident in the matrix of NFB with fibrous CT and inflammatory cells on the advancing edge of NFB. Blue arrows show the boundary between NFB and NB (×40). BV, bone volume; CM, collagen membrane; CR, CSD, calvaria critical size defect; CT, connective tissue; NB, native bone; NFB, newly formed bone.

Figure 4

Histologic assessment (using hematoxylin-eosin staining) of CSD and native calvaria in group 2. (a) Edges of the CSD (red arrows) bridged by a thin strip of NFB extending from one edge of the CSD (×4). (b) NFB seen on the edge and in the middle of the CSD with CT fibers bridging the NFB with the edge of the CSD. Remnants of CM can be seen as an acellular acidophilic material with no evidence of inflammation (×10). (c) Osteocytes (green arrows) were evident in the matrix of NFB with fibrous CT adjacent to the leading edge of the NFB. There is no evidence of inflammation and remnants of CM were visible as an acellular acidophilic material (×40). BV, bone volume; CM, collagen membrane; CR, CSD, calvaria critical size defect; CT, connective tissue; NB, native bone; NFB, newly formed bone.

Figure 5

Histologic assessment (using hematoxylin-eosin staining) of CSD and native calvaria in group 3. (a) Edges of the CSD (red arrows) bridging the NFB with a granular crystalline material interspersed with fibrous CT (×4). (b) Blue arrows show the boundary between the NFB and NB with remnants of particulate graft interspersed with fibrous CT and encapsulated by inflammatory cells (black arrows) (×10). (c) Osteocytes (green arrows) were visible in the matrix of NFB remaining bone graft particles seen as a granular crystalline material along with inflammatory encapsulation (black arrows) (×40). BV, bone volume; CM, collagen membrane; CR, CSD, calvaria critical size defect; CT, connective tissue; NB, native bone; NFB, newly formed bone; TCP, tricalcium phosphate.

Figure 6

Load displacement curves and calculated hardness values (H) generated from nanoindentation of calvarial bone of a rat. (a) Natural bone adjacent to the defect site; (b) regenerated bone with BMSC, TCP, and membrane; (c) regenerated bone with BMSC and membrane. BMSC, bone marrow-derived mesenchymal stem cell; TCP, tricalcium phosphate.

Figure 7

In situ SPM image of residual indent showing residual impression/or fracture events. This image was taken using the same indentation tip that performed the testing. (a) Natural adjacent bone to the calvarial defect; (b) regenerated bone following the use of BMSC, TCP, and membrane; (c) regenerated bone with BMSCs and membrane. BMSC, bone marrow derived mesenchymal stem cell; SPM, scanning probe microscopy; TCP, tricalcium phosphate.