BMP2 is superior to BMP4 for promoting human muscle-derived stem cell-mediated bone regeneration in a critical-sized calvarial defect model

Abstract

Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is more efficient than BMP4 in promoting the bone regenerative capacity of the hMDSCs in vivo.

Figure 1

Characterization of the human muscle-derived stem cells (hMDSCs). (A) Flow cytometry analysis of hMDSC surface markers revealed robust expression of cluster of differentiation 56 (CD56), CD146, CD44, CD73, CD90, and CD105 but not CD45 or Ulexeuropaeus agglutinin I receptor (UEA-1R). (B) Quantification of cell lineage marker expression in hMDSCs from four donors demonstrated consistency in their cell marker expression profiles. (C) RT-PCR results of stem cell markers. Cells from each donor expressed the stem cell markers octamer-binding transcription factor 4 (OCT4) and NANOG, but only a faint expression of sex-determining region Y box-2 (SOX-2) was detected in cells from two donors (3 and 4). Glyceraldehyde 3-phosphate dehydroge-nase (GAPDH) was used as a loading control. (D) Multipotent differentiation of hMDSCs. hMDSCs undergo adipogenesis (oil red staining), chondrogenesis (Alcian blue staining), osteogenesis (von Kossa staining), and myogenesis as shown by fast myosin heavy chain (fMHC) and desmin immunofluorescence. Scale bars: 200 µm.

Figure 2

Osteogenic differentiation of hMDSCs and retro-BMP2/4GFP-transduced hMDSCs in vitro. (A) Schematic illustration of the retro-bone morphogenetic protein 2/4 green fluorescent protein (BMP2/4GFP) vector. (B) The 100% GFP-positive retro-BMP2/4GFP-transduced hMDSCs were obtained by fluorescence-activated cell sorting (FACS). Scale bars: 200 µm. (C) RT-PCR results indicated no significant gene expression changes before and after retro-BMP2/4GFP transduction of the hMDSCs. (D) Microcomputed tomography (microCT) evaluation of matrix mineralization of retro-BMP2/4GFP-transduced and nontransduced hMDSCs in the osteogenic pellet culture assay. **p < 0.01 compared to nontransduced hMDSCs (n = 4). (E) von Kossa staining of the pellets at 4 weeks showed that both transduced and nontransduced hMDSCs underwent osteogenesis. Osteocalcin staining indicated the differentiation of hMDSCs into osteogenic lineage. Scale bars: 100 µm. LTR, long terminal repeat; CMV, cytomegalovirus; IRES, internal ribosome entry site; BMPR1B, bone morphogenetic protein receptor 1B; COX-2, cyclooxygenase-2; mg HA/cm³, mg hydroxyapatite per cubic cm.

Figure 3

Bone regeneration after transplantation of retro-BMP2/4GFP-transduced hMDSCs. (A) No bone formation was detected by microCT within the defect area at 42 days after the transplantation of retro-BMP2/4GFP-transduced hMDSCs using either fibrin sealant (FS) or Gelfoam (GF) in B6 severe combined immunodeficient (SCID) mice. Complete defect healing was observed 4 weeks after the transplantation of retro-BMP4GFP-transduced mMDSCs (mMDSC + FS). (B) No bone regeneration was observed after transplantation of BMP4 stimulation (+BMP4) or NAC treatment plus BMP4 (+BMP4+NAC) retro-BMP2/4GFP-transduced hMDSCs in CBSCBG SCID (SCID beige) mice at any time point. (C) Many GFP-positive donor cells (green), granulocyte receptor-1 (Gr-1)-positive host neutrophils (red), and vascular endothelial cells (red) were found at the defect site 1 week postimplantation, but only a very few GFP-positive cells were detected after 3 weeks. Only isolated spots of mineralization were identified in the defect area as shown by von Kossa staining (black) at 21 days. Scale bar: 100 µm. DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride.

Figure 4

The effect of BMP4 protein stimulation on hMDSC proliferation in vitro. (A) hMDSC proliferation increased after 3 days when 50 or 100 ng/ml of BMP4 was added to the culture medium on day 1. (B) hMDSC proliferation decreased after 5 days when BMP4 was added on days 1 and 3 using three different dosages (25, 50, or 100 ng/ml). *p < 0.05, **p < 0.01 compared to the untreated controls. n = 4 in each treatment group. (C) hMDSCs did not express alkaline phosphatase (AP) after BMP4 stimulation at 3 or 5 days. Scale bars: 200 µm. (D) C2C12 cells expressed AP after BMP4 stimulation for 3 days. Scale bars: 200 µm. dsDNA, double-stranded DNA.

Figure 5

BMP2 secretion levels and in vitro multipotent differentiation of hMDSC after lenti-BMP2 transduction. (A) Schematic illustration of the lenti-BMP2 vector. Red arrows indicate insertion sites of the target BMP2 gene. (B) BMP2 secretion after hMDSC transduction and selection with blasticidin at passages 1, 2, and 5. (C) AP staining showed no increase in AP-positive cell (arrow) number after hMDSC transduction with lenti-BMP2. (D) Osteogenic differentiation was enhanced after transduction of hMDSCs with lenti-BMP2 as shown by von Kossa staining, osteocalcin immunohistochemistry, and microCT. (E) Lenti-BMP2 transduction enhanced chondrogenic differentiation but prevented from myogenic differentiation. (F) RT-PCR indicated no significant change in osteogenic-related genes after lenti-BMP2 transduction. Scale bars: 200 µm. BLP, passage after blasticidin selection.

Figure 6

Bone regeneration after transplantation of lenti-BMP2-transduced hMDSCs. (A) MicroCT images of the different treatment groups showed negligible bone formation in the retro-BMP2/4GFP-transduced hMDSCs (hMDSC-retro-BMP2/4GFP) and scaffold control groups. In contrast, robust bone formation was observed in the lenti-BMP2-transduced hMDSC (hMDSC-lenti-BMP2) group at 2 weeks, and nearly complete defect healing was detected by 6 weeks. (B) Quantification of the calvarial defect area covered by the regenerated bone after 6 weeks showed 80%, 10%, and 14% healing in the hMDSC-lenti-BMP2 group, hMDSC-retro-BMP2/4GFP, and scaffold control groups, respectively. (C) Quantification of the regenerated bone volume after 6 weeks showed significantly more bone in the hMDSC-lenti-BMP2 group compared to the other groups, **p < 0.01, compared to scaffold control, ^##p < 0.01, compared to the hMDSC-retro-BMP2/4GFP group.

Figure 7

Morphometric and histological characteristics of the newly regenerated calvarial bone. (A) MicroCT demonstrated that the regenerated bone in the hMDSC-lenti-BMP2 group was similar to the native bone and was well integrated into the surrounding host bone. (B) Herovici’s staining of cross sections of skull tissues (20×) showing that the regenerated bone (the area between arrows indicate calvarial defect region) in the hMDSC-lenti-BMP2 group was similar to the native bone in the contralateral side (area between stars). (C) Higher magnification (200×) of the regenerated bone area (box area in B) showing that hMDSC-lenti-BMP2 group contained mainly type I collagen (red) and was well integrated with the host bone (arrow). The tissue within the defect in scaffold control and hMDSC-retro-BMP2/4GFP groups mainly consisted of type III collagen (blue). Scale bars: 100 µm. COL1, type I collagen; COL3, type III collagen; Br, brain; BM, bone marrow.