Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular "Critical Size" Defect

Abstract

Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs.

Figure 1

(a) In vitro appearance of BMSSCs forming a matrix nodule after two weeks of culture in the presence of osteoblast differentiation medium. (b) Cultures of BMSSCs stained for ALP expression. (c) In vitro appearance of BMSSCs forming a matrix nodule in proximity of BPB before grafting.

Figure 2

Three critical-size circular defects. Clinical situation during surgery.

Figure 3

Standard RX 55 × 75 mm films have been performed on retrieved mandibulae to find the perfect position and alignment of biomaterial scaffolds with the bone itself.

Figure 4

(a) Control group. Trabecular bone (TB) was present in the central portion of the bone defects (c). New bone (arrows) extended to the basal third from the margin of the bone defect. Acid fuchsin and toluidine blue. Bar = 200 μ. (b) Previous image at higher magnification. Isolated trabecular bone (TB was seen throughout the medullary space (MS). Acid fuchsin and toluidine blue. Bar = 200 μ.

Figure 5

(a) BPB group. New bone surrounded the block material (BPB). New bone (arrows) extended also in the central part of the bone defects. Acid fuchsin and toluidine. Bar = 200 μ. (b) BPB/BMSSCs group. New bone (arrows) was deposited in the block material (BPB). No fibrous tissue was observed in the defect area. Acid fuchsin and toluidine. Bar = 200 μ.

Figure 6

(a) BPB group. Bone tissue was deposited in the block material (BPB). No fibrous tissue was observed in the defect area. Vessels (V) were present in the central part of the bone defects. New bone (NB) and osteoblasts in close contact with the block material acid fuchsin and toluidine blue. Bar = 100 μ. (b) BPB/BMSSCs group. Mature bone (MB) in the bone defect. Bone morphology was more mature and well organized, presenting a primary osteon. A basic multicellular unit of osteoclasts cells (yellow arrows) that dissolves an area of the bone surface and then fills it with new bone by osteoblasts (white arrows) to form haversian systems or osteons. A vessels (V) were present in the central part of the bone defects. Acid fuchsin and toluidine blue. Bar = 100 μ.

Figure 7

Percentage bar graphic showing difference between site compositions in three groups: control, bone porcine block, and bone marrow stromal stem cells/bone porcine block.