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Efficacy of autologous stem cell-based therapy for osteonecrosis of the femoral head in sickle cell disease: a five-year follow-up study.

Daltro GC, Fortuna V, de Souza ES, Salles MM, Carreira AC, Meyer R, Freire SM, Borojevic R - Stem Cell Res Ther (2015)

Bottom Line: Flow cytometry analysis showed a significantly higher number of hematopoietic stem/endothelial progenitor cell markers in concentrated BMMCs when compared with bone marrow aspirate, indicating an enrichment of these cell types.The autologous BMMC implantation with a minimally invasive technique resulted in significant pain relief and halted the progression of early stages of ONFH in SCD patients.In summary, our results indicate that infusion of BMMCs enriched with stem/progenitor cells is a safe and effective treatment for the early stages of ONFH in SCD patients.

View Article: PubMed Central - PubMed

Affiliation: Prof. Edgar Santos Hospital Complex, Salvador, BA, 40110-902, Brazil. gildasio.daltro@yahoo.com.br.

ABSTRACT

Introduction: Stem cell therapy with bone marrow-derived mononuclear cells (BMMCs) is an option for improving joint function in osteonecrosis of the femoral head (ONFH). Bone marrow-derived mesenchymal stromal cell (MSC) numbers and their osteogenic differentiation are decreased in patients with ONFH. However, whether this decrease also extends to the early stages of ONFH in sickle cell disease (SCD) is still unclear.

Methods: We conducted a phase I/II, non-controlled study to determine efficacy and safety of BMMC implantation using a minimally invasive technique in SCD patients with ONFH. Eighty-nine patients were recruited and followed up for 60 months after surgery. Clinical and radiographic findings were assessed, and data were completed by in vitro analysis.

Results: At the final follow-up (60 months) there was a significant improvement in clinical joint symptoms and pain relief as measured by the Harris Hip Score (P = 0.0005). In addition, after the BMMC implantation procedure, radiographic assessment showed disease stabilization and only 3.7 % of the treated patients did not achieve a satisfactory clinical result. The amount of fibroblast colony-forming units was 28.2 ± 13.9 per 1 million BMMCs after concentration. Flow cytometry analysis showed a significantly higher number of hematopoietic stem/endothelial progenitor cell markers in concentrated BMMCs when compared with bone marrow aspirate, indicating an enrichment of these cell types. Isolated MSCs from SCD patients with pre-collapse ONFH maintained the replicative capacity without significant loss of their specific biomolecular characteristics, multi-differentiation potential, and osteogenic differentiation activities. Cytokines and growth factors (interleukin-8, transforming growth factor-beta, stromal cell-derived factor-1alpha and vascular endothelial growth factor) that mediate endogenous bone regeneration were also produced by expanded MSCs from SCD patients.

Conclusion: The autologous BMMC implantation with a minimally invasive technique resulted in significant pain relief and halted the progression of early stages of ONFH in SCD patients. MSCs from SCD patients display biological properties that may add to the efficiency of surgical treatment in ONFH. In summary, our results indicate that infusion of BMMCs enriched with stem/progenitor cells is a safe and effective treatment for the early stages of ONFH in SCD patients.

Trial registration: ClinicalTrials.gov NCT02448121; registered 15 May 2015.

No MeSH data available.


Related in: MedlinePlus

Osteogenic abilities of bone marrow-derived MSCs are not defective in patients with osteonecrosis. a Quantitative RT-PCR of osteoblast-related genes from control and osteogenic-induced MSCs after 10 days were normalized and compared according to housekeeping genes as control. b Scatter plots (left) showing the distribution of total fluorescence intensity from FACS analysis (right) of cellular osteocalcin expression in MSCs. The relative fluorescence intensity shows significant differences between control and osteogenic differentiation conditions. c Alizarin Red staining (left) indicating mineralized nodule formation of cultured MSCs after treatment with osteogenic media for 21 days. Quantitative analysis of the amount of Alizarin staining in each group is also indicated (right). No significant difference (NS) was found between MSCs from SCD patients with early-stage osteonecrosis and patients with further stages of the disease at a distant site. Alizarin Red staining was independent of the disease stage. d Enzyme-linked immunosorbent assay for interleukin-8 (IL-8), transforming growth factor-beta (TGF-β), stromal cell-derived factor-1alpha (SDF-1α) and vascular endothelial growth factor (VEGF). Concentration (pg/mL) of cytokines and growth factors in supernatants of MSCs of SCD patients after 2 days of culture. MSCs from SCD patients produce distinct levels of IL-8. Horizontal bars represent the mean and SD of data. The triangles represent individual values. *P < 0.05, **P < 0.01, ***P < 0.001
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Fig6: Osteogenic abilities of bone marrow-derived MSCs are not defective in patients with osteonecrosis. a Quantitative RT-PCR of osteoblast-related genes from control and osteogenic-induced MSCs after 10 days were normalized and compared according to housekeeping genes as control. b Scatter plots (left) showing the distribution of total fluorescence intensity from FACS analysis (right) of cellular osteocalcin expression in MSCs. The relative fluorescence intensity shows significant differences between control and osteogenic differentiation conditions. c Alizarin Red staining (left) indicating mineralized nodule formation of cultured MSCs after treatment with osteogenic media for 21 days. Quantitative analysis of the amount of Alizarin staining in each group is also indicated (right). No significant difference (NS) was found between MSCs from SCD patients with early-stage osteonecrosis and patients with further stages of the disease at a distant site. Alizarin Red staining was independent of the disease stage. d Enzyme-linked immunosorbent assay for interleukin-8 (IL-8), transforming growth factor-beta (TGF-β), stromal cell-derived factor-1alpha (SDF-1α) and vascular endothelial growth factor (VEGF). Concentration (pg/mL) of cytokines and growth factors in supernatants of MSCs of SCD patients after 2 days of culture. MSCs from SCD patients produce distinct levels of IL-8. Horizontal bars represent the mean and SD of data. The triangles represent individual values. *P < 0.05, **P < 0.01, ***P < 0.001

Mentions: The immunocytochemical staining results for osteogenic induction were confirmed by quantitative PCR. Osteoblast lineage-specific markers (RUNX2, ALPL and COL1) are expressed during in vitro osteodifferentiation of MSCs and precede the late-stage mineralization process [36, 37]. After 10 days of differentiation, significantly higher expression levels of osteogenic specific genes RUNX2, ALPL and COL1 were observed in MSCs cultured under osteogenic medium, as compared with control MSC samples from at least five SCD patients (Fig. 6a). Quantitative PCR analysis showed that mRNA fold-changes of the osteogenic genes were highly similar in differentiated MSCs, with the exception of ENG mRNA expression levels, which were similar between control and osteogenic-induced samples (Fig. 6a). Expressions of genes specific for osteogenesis were not significantly different between samples from HbSS and HbSC. Osteocalcin, a marker of developing osteoblasts, was detected at higher levels in osteogenic-induced MSCs compared to control MSCs after 10 days (Fig. 6b). The upregulation of osteogenesis-specific genes had already started after 10 days of differentiation, although no calcium formation was observed at this time point. However, after 21 days, the mean percentage of the area stained with Alizarin Red S was 28.3 ± 10.5 % in MSCs cultured under osteogenic medium, as compared with control MSC samples from SCD patients with ONFH. The amount of mineralized matrix deposited during in vitro osteogenic differentiation was not statistically different among MSCs of SCD patients with pre-collapse osteonecrosis stages (Fig. 6c). These results indicated that ex vivo MSCs isolated from SCD patients maintain the ability of osteogenic differentiation after population expansion in our culture conditions.Fig. 6


Efficacy of autologous stem cell-based therapy for osteonecrosis of the femoral head in sickle cell disease: a five-year follow-up study.

Daltro GC, Fortuna V, de Souza ES, Salles MM, Carreira AC, Meyer R, Freire SM, Borojevic R - Stem Cell Res Ther (2015)

Osteogenic abilities of bone marrow-derived MSCs are not defective in patients with osteonecrosis. a Quantitative RT-PCR of osteoblast-related genes from control and osteogenic-induced MSCs after 10 days were normalized and compared according to housekeeping genes as control. b Scatter plots (left) showing the distribution of total fluorescence intensity from FACS analysis (right) of cellular osteocalcin expression in MSCs. The relative fluorescence intensity shows significant differences between control and osteogenic differentiation conditions. c Alizarin Red staining (left) indicating mineralized nodule formation of cultured MSCs after treatment with osteogenic media for 21 days. Quantitative analysis of the amount of Alizarin staining in each group is also indicated (right). No significant difference (NS) was found between MSCs from SCD patients with early-stage osteonecrosis and patients with further stages of the disease at a distant site. Alizarin Red staining was independent of the disease stage. d Enzyme-linked immunosorbent assay for interleukin-8 (IL-8), transforming growth factor-beta (TGF-β), stromal cell-derived factor-1alpha (SDF-1α) and vascular endothelial growth factor (VEGF). Concentration (pg/mL) of cytokines and growth factors in supernatants of MSCs of SCD patients after 2 days of culture. MSCs from SCD patients produce distinct levels of IL-8. Horizontal bars represent the mean and SD of data. The triangles represent individual values. *P < 0.05, **P < 0.01, ***P < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig6: Osteogenic abilities of bone marrow-derived MSCs are not defective in patients with osteonecrosis. a Quantitative RT-PCR of osteoblast-related genes from control and osteogenic-induced MSCs after 10 days were normalized and compared according to housekeeping genes as control. b Scatter plots (left) showing the distribution of total fluorescence intensity from FACS analysis (right) of cellular osteocalcin expression in MSCs. The relative fluorescence intensity shows significant differences between control and osteogenic differentiation conditions. c Alizarin Red staining (left) indicating mineralized nodule formation of cultured MSCs after treatment with osteogenic media for 21 days. Quantitative analysis of the amount of Alizarin staining in each group is also indicated (right). No significant difference (NS) was found between MSCs from SCD patients with early-stage osteonecrosis and patients with further stages of the disease at a distant site. Alizarin Red staining was independent of the disease stage. d Enzyme-linked immunosorbent assay for interleukin-8 (IL-8), transforming growth factor-beta (TGF-β), stromal cell-derived factor-1alpha (SDF-1α) and vascular endothelial growth factor (VEGF). Concentration (pg/mL) of cytokines and growth factors in supernatants of MSCs of SCD patients after 2 days of culture. MSCs from SCD patients produce distinct levels of IL-8. Horizontal bars represent the mean and SD of data. The triangles represent individual values. *P < 0.05, **P < 0.01, ***P < 0.001
Mentions: The immunocytochemical staining results for osteogenic induction were confirmed by quantitative PCR. Osteoblast lineage-specific markers (RUNX2, ALPL and COL1) are expressed during in vitro osteodifferentiation of MSCs and precede the late-stage mineralization process [36, 37]. After 10 days of differentiation, significantly higher expression levels of osteogenic specific genes RUNX2, ALPL and COL1 were observed in MSCs cultured under osteogenic medium, as compared with control MSC samples from at least five SCD patients (Fig. 6a). Quantitative PCR analysis showed that mRNA fold-changes of the osteogenic genes were highly similar in differentiated MSCs, with the exception of ENG mRNA expression levels, which were similar between control and osteogenic-induced samples (Fig. 6a). Expressions of genes specific for osteogenesis were not significantly different between samples from HbSS and HbSC. Osteocalcin, a marker of developing osteoblasts, was detected at higher levels in osteogenic-induced MSCs compared to control MSCs after 10 days (Fig. 6b). The upregulation of osteogenesis-specific genes had already started after 10 days of differentiation, although no calcium formation was observed at this time point. However, after 21 days, the mean percentage of the area stained with Alizarin Red S was 28.3 ± 10.5 % in MSCs cultured under osteogenic medium, as compared with control MSC samples from SCD patients with ONFH. The amount of mineralized matrix deposited during in vitro osteogenic differentiation was not statistically different among MSCs of SCD patients with pre-collapse osteonecrosis stages (Fig. 6c). These results indicated that ex vivo MSCs isolated from SCD patients maintain the ability of osteogenic differentiation after population expansion in our culture conditions.Fig. 6

Bottom Line: Flow cytometry analysis showed a significantly higher number of hematopoietic stem/endothelial progenitor cell markers in concentrated BMMCs when compared with bone marrow aspirate, indicating an enrichment of these cell types.The autologous BMMC implantation with a minimally invasive technique resulted in significant pain relief and halted the progression of early stages of ONFH in SCD patients.In summary, our results indicate that infusion of BMMCs enriched with stem/progenitor cells is a safe and effective treatment for the early stages of ONFH in SCD patients.

View Article: PubMed Central - PubMed

Affiliation: Prof. Edgar Santos Hospital Complex, Salvador, BA, 40110-902, Brazil. gildasio.daltro@yahoo.com.br.

ABSTRACT

Introduction: Stem cell therapy with bone marrow-derived mononuclear cells (BMMCs) is an option for improving joint function in osteonecrosis of the femoral head (ONFH). Bone marrow-derived mesenchymal stromal cell (MSC) numbers and their osteogenic differentiation are decreased in patients with ONFH. However, whether this decrease also extends to the early stages of ONFH in sickle cell disease (SCD) is still unclear.

Methods: We conducted a phase I/II, non-controlled study to determine efficacy and safety of BMMC implantation using a minimally invasive technique in SCD patients with ONFH. Eighty-nine patients were recruited and followed up for 60 months after surgery. Clinical and radiographic findings were assessed, and data were completed by in vitro analysis.

Results: At the final follow-up (60 months) there was a significant improvement in clinical joint symptoms and pain relief as measured by the Harris Hip Score (P = 0.0005). In addition, after the BMMC implantation procedure, radiographic assessment showed disease stabilization and only 3.7 % of the treated patients did not achieve a satisfactory clinical result. The amount of fibroblast colony-forming units was 28.2 ± 13.9 per 1 million BMMCs after concentration. Flow cytometry analysis showed a significantly higher number of hematopoietic stem/endothelial progenitor cell markers in concentrated BMMCs when compared with bone marrow aspirate, indicating an enrichment of these cell types. Isolated MSCs from SCD patients with pre-collapse ONFH maintained the replicative capacity without significant loss of their specific biomolecular characteristics, multi-differentiation potential, and osteogenic differentiation activities. Cytokines and growth factors (interleukin-8, transforming growth factor-beta, stromal cell-derived factor-1alpha and vascular endothelial growth factor) that mediate endogenous bone regeneration were also produced by expanded MSCs from SCD patients.

Conclusion: The autologous BMMC implantation with a minimally invasive technique resulted in significant pain relief and halted the progression of early stages of ONFH in SCD patients. MSCs from SCD patients display biological properties that may add to the efficiency of surgical treatment in ONFH. In summary, our results indicate that infusion of BMMCs enriched with stem/progenitor cells is a safe and effective treatment for the early stages of ONFH in SCD patients.

Trial registration: ClinicalTrials.gov NCT02448121; registered 15 May 2015.

No MeSH data available.


Related in: MedlinePlus