INTRODUCTION: Current therapeutic regimens in osteoarthritis (OA) address mainly pain but not the slow progressive degradation of the extracellular matrix (ECM) and the loss of a chondrogenic phenotype in articular cartilage. In the present study, using an early OA cancellous bone scaffold, we aimed to uncover evidence of the successful hyaline cartilage regenerative capacity of autologous human granulocyte colony-stimulating factor (hG-CSF)-activated peripheral blood stem cells (AAPBSC) with growth factor addition. MATERIALS AND METHODS: AAPBSC were harvested in ten patients (median age 58 years, 8 females), and flow cytometry was performed for cell surface markers. Arthroscopically obtained cancellous bone scaffold specimens were seeded with AAPBSC. In Group 1, the scaffold was seeded with AAPBSC only, in Group 2, AAPBSC plus hyaluronic acid (HA), and in Group 3, AAPBSC plus HA, hG-CSF, and double-centrifuged platelet-rich plasma (PRP). The specimens were analyzed for cell attachment and proliferation by the fluorometric quantification of cellular DNA assay and scanning electron microscopy. Chondrogenic gene expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of Sox9, collagen type II (COL-2), and aggrecan. Histological sections of scaffold constructs for cartilaginous matrix formation were stained with toluidine blue (proteoglycan) and safranin O (sGAG) after 3 weeks. RESULTS: AAPBSC displayed especially high levels of CD29 and CD44 surface markers, as well as CD90, and CD105, while only a small proportion expressed CD34. Almost half of the seeded cells attached on the bone scaffolds in all three groups (not statistically significant), whereas the means of cell proliferation on day 7 compared to day 1 were statistically significant difference with the order of increase as group 3 > group 2 > group 1. RT-PCR showed statistically significant sequential increases in Sox9, COL-2, and Aggrecan all being highest in group 3. Histological analysis demonstrated cells in the cancellous bone scaffold with a round morphology, and ECM was positively stained by toluidine blue and safranin O indicating increased proteoglycan and glycosaminoglycan content, respectively, in the newly formed cartilage matrix. CONCLUSIONS: AAPBSC initiated chondrocyte differentiation on an autologous cancellous bone scaffold, and the addition of PRP and hG-CSF further stimulated cell proliferation toward a chondrocyte phenotype with potentiated Sox9 transcription resulting in sequential COL-2 and aggrecan mRNA increases that ultimately resulted in histologically confirmed increased proteoglycan and glucosaminoglycan content in newly formed hyaline cartilage.
INTRODUCTION: Current therapeutic regimens in osteoarthritis (OA) address mainly pain but not the slow progressive degradation of the extracellular matrix (ECM) and the loss of a chondrogenic phenotype in articular cartilage. In the present study, using an early OA cancellous bone scaffold, we aimed to uncover evidence of the successful hyaline cartilage regenerative capacity of autologous humangranulocyte colony-stimulating factor (hG-CSF)-activated peripheral blood stem cells (AAPBSC) with growth factor addition. MATERIALS AND METHODS: AAPBSC were harvested in ten patients (median age 58 years, 8 females), and flow cytometry was performed for cell surface markers. Arthroscopically obtained cancellous bone scaffold specimens were seeded with AAPBSC. In Group 1, the scaffold was seeded with AAPBSC only, in Group 2, AAPBSC plus hyaluronic acid (HA), and in Group 3, AAPBSC plus HA, hG-CSF, and double-centrifuged platelet-rich plasma (PRP). The specimens were analyzed for cell attachment and proliferation by the fluorometric quantification of cellular DNA assay and scanning electron microscopy. Chondrogenic gene expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) of Sox9, collagen type II (COL-2), and aggrecan. Histological sections of scaffold constructs for cartilaginous matrix formation were stained with toluidine blue (proteoglycan) and safranin O (sGAG) after 3 weeks. RESULTS: AAPBSC displayed especially high levels of CD29 and CD44 surface markers, as well as CD90, and CD105, while only a small proportion expressed CD34. Almost half of the seeded cells attached on the bone scaffolds in all three groups (not statistically significant), whereas the means of cell proliferation on day 7 compared to day 1 were statistically significant difference with the order of increase as group 3 > group 2 > group 1. RT-PCR showed statistically significant sequential increases in Sox9, COL-2, and Aggrecan all being highest in group 3. Histological analysis demonstrated cells in the cancellous bone scaffold with a round morphology, and ECM was positively stained by toluidine blue and safranin O indicating increased proteoglycan and glycosaminoglycan content, respectively, in the newly formed cartilage matrix. CONCLUSIONS: AAPBSC initiated chondrocyte differentiation on an autologous cancellous bone scaffold, and the addition of PRP and hG-CSF further stimulated cell proliferation toward a chondrocyte phenotype with potentiated Sox9 transcription resulting in sequential COL-2 and aggrecan mRNA increases that ultimately resulted in histologically confirmed increased proteoglycan and glucosaminoglycan content in newly formed hyaline cartilage.
Authors: Antonio Marmotti; Roberto Rossi; Filippo Castoldi; Eliana Roveda; Gianni Michielon; Giuseppe M Peretti Journal: Biomed Res Int Date: 2015-05-05 Impact factor: 3.411