Bone Progenitors Produced by Direct Osteogenic Differentiation of the Unprocessed Bone Marrow Demonstrate High Osteogenic PotentialIn VitroandIn Vivo

Abstract

Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×106. In comparison, the yield of MSCs produced by adhesion of mononuclear cells derived from the same amount of BM and cultured in a commercial growth medium for 2 weeks was 0.3±0.17×106. When a scaffold was added to the BM-OM mixture, and the mixture was cultured in a simple rotational bioreactor; the resulting BPCs were obtained already seeded on the scaffold. BPCs seeded on scaffolds were capable of proliferation for at least 6 weeks, keeping high levels of ALP activity, expressing osteogenic genes, and mineralizing the scaffolds. Autologous rat BPCs seeded on various scaffolds were transplanted into critical-size calvarial defects. Six weeks after transplantation of polylactic acid/polyglycolic acid scaffolds, 76.1%±18.3% of the defects were filled with a new bone, compared to 37.9%±28.4% in the contralateral defects transplanted with the scaffolds without cells.

Identification of Gli1‐interacting proteins during simvastatin‐stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

“…The phenotype of BMSCs includes negative expression of CD11b and CD45 cell surface markers, as confirmed by flow cytometry . Successful osteogenic differentiation of BMSCs is demonstrated by increased ALP staining and enhanced matrix mineralization …”

Changes of Bone Turnover Markers in Long Bone Nonunions Treated with a Regenerative Approach

“…Following any MSC-based therapeutic intervention on bone, the amount of tissue that is regenerated cannot be reliably predicted. As remarked by Ginis et al, radiological images may overestimate the extent of bone formation during regeneration, but the identification of “true” new bone at the site of regeneration, as obtained by histology and histomorphometry in “in vivo” models, is not feasible in humans [ 29 ]. Biomarkers of the activity of bone remodeling may provide additional information beyond radiographic assessments, but to date, a biological factor useful to the clinical monitoring of bone healing has not been identified [ 30 ].…”

Osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells encapsulated in Thai silk fibroin/collagen hydrogel: a pilot study in vitro

“…p-Nitrophenyl phosphate (100 mL) was added to the cell lysates in each well and then together incubated at 37°C under an atmosphere of 5% CO 2 for 15 min. The reaction was stopped with 80 mL of 0.02 N NaOH, and p-nitrophenol, which is a product of hydrolysis of p-nitrophenol phosphate catalyzed by ALP, was measured using a microplate reading spectrophotometer at 405 nm [20].…”