Optimization of an ex vivo gene transfer to the hamstrings tendons muscle remnants: potential for genetic enhancement of bone healing.

AIM: To assess whether an adenoviral vector carrying the bone morphogenetic protein genes (Ad.BMP-2) can transduce human muscle tissue and direct it toward osteogenic differentiation within one hour. METHODS: This in vitro study, performed at the Department of Molecular Biology, Faculty of Science, Zagreb from 2012 to 2017, used human muscle tissue samples collected during anterior cruciate ligament reconstructions performed in St Catherine Hospital, Zabok. Samples from 28 patients were transduced with adenoviral vector carrying firefly luciferase cDNA (Ad.luc) by using different doses and times of transduction, and with addition of positive ions for transduction enhancement. The optimized protocol was further tested on muscle samples from three new patients, which were transduced with Ad.BMP-2. Released bone morphogenetic protein 2 (BMP-2) levels in osteogenic medium were measured every three days during a period of 21 days. Expression of osteogenic markers was measured at day 14 and 21. After 21 days of cultivation, muscle tissue was immunohistochemically stained for collagen type I detection (COL-I). RESULTS: The new transduction protocol was established using 108 plaque-forming units (P<0.001) as an optimal dose of adenoviral vector and 30 minutes (P<0.001) as an optimal contact time. Positive ions did not enhance transduction. Samples transduced with Ad.BMP-2 according to the optimized protocol showed enhanced expression of osteogenic markers (P<0.050), BMP-2 (P<0.001), and COL I. CONCLUSION: This study confirms that Ad.BMP-2 can transduce human muscle tissue and direct it toward osteogenic differentiation within 30 minutes.

FADD-deficient mouse embryonic fibroblasts undergo RIPK1-dependent apoptosis and autophagy after NB-UVB irradiation.

Sun or therapy-related ultraviolet B (UVB) irradiation induces different cell death modalities such as apoptosis, necrosis/necroptosis and autophagy. Understanding of mechanisms implicated in regulation and execution of cell death program is imperative for prevention and treatment of skin diseases. An essential component of death-inducing complex is Fas-associated protein with death domain (FADD), involved in conduction of death signals of different death modalities. The purpose of this study was to enlighten the role of FADD in the selection of cell death mode after narrow-band UVB (NB-UVB) irradiation using specific cell death inhibitors (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (zVAD-fmk), Necrostatin-1 and 3-Methyladenine) and FADD-deficient (FADD-/-) mouse embryonic fibroblasts (MEFs) and their wild type (wt) counterparts. The results imply that lack of FADD sensitized MEFs to induction of receptor-interacting protein 1 (RIPK1)-dependent apoptosis by the generation of reactive oxygen species (ROS), but without activation of the proteins p53, Bax and Bcl-2 as well as without the enrolment of calpain-2. Autophagy was established as a contributing factor to NB-UVB-induced death execution. By contrast, wt cells triggered intrinsic apoptotic pathway that was resistant to the inhibition by zVAD-fmk and Necrostatin-1 pointing to the mechanism overcoming the cell survival. These findings support the role of FADD in prevention of autophagy-dependent apoptosis.

Cellular hydrogels based on pH-responsive chitosan-hydroxyapatite system.

The development of bioactive injectable system as cell carrier with minimal impact on viability of encapsulated cells represents a great challenge. In the present work, we propose a new pH-responsive chitosan-hydroxyapatite-based hydrogel with sodium bicarbonate (NaHCO3) as the gelling agent. The in situ synthesis of hydroxyapatite phase has resulted in stable composite suspension and final homogeneous hydrogel. The application of sodium bicarbonate has allowed non-cytotoxic fast gelation of chitosan-hydroxyapatite within 4min, and without excess of sodium ions concentration. Rheological properties of crosslinked hydrogel have demonstrated possible behaviour as 'strong physical hydrogel'. The live dead staining has confirmed good viability and dispersion, as well as proliferation of encapsulated cells by the culture time. Presented preliminary results show good potential of chitosan-hydroxyapatite/NaHCO3 as a cell carrier, whose impact on the cell differentiation need to be confirmed by encapsulation of other cell phenotypes.

Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction.

PURPOSE: The aim of this study is to examine the capacity of muscle tissue preserved on hamstring tendons forming candy-stripe grafts in order to improve tendon to bone ingrowth and ligamentization. We hypothesized that muscle tissue does possess a stem cell population that could enhance the healing process of the ACL graft when preserved on the tendons. METHODS: Human samples from gracilis and semitendinosus muscles were collected during ACL surgery from ten patients and from these tissue samples human muscle-derived stem cells and tendon-derived stem cells were isolated and propagated. Both stem cell populations were in-vitro differentiated into osteogenic lineage. Alkaline phosphatase activity was determined at days zero and 14 of the osteogenic induction and von Kossa staining to assess mineralization of the cultures. Total RNA was collected from osteoblast cultures and real time quantitative PCR was performed. Western-blot for osteocalcin and collagen type I followed protein isolation. Immunofluorescence double labeling of pericytes in muscle and tendon tissue was performed. RESULTS: Mesenchymal stem cells from muscle and tendon tissue were isolated and expanded in cell culture. More time was needed to grow the tendon derived culture compared to muscle derived culture. Muscle derived stem cells exhibited more alkaline phosphatase actvity compared to tendon derived stem cells, whereas tendon derived stem cells formed more mineralized nodules after 14 days of osteoinduction. Muscle derived stem cells exhibited higher expression levels of bone sialoprotein, and tendon derived stem cells showed higher expression of dental-matrix-protein 1 and osteocalcin. Immunofluorescent staining against pericytes indicated that they are more abundant in muscle tissue. CONCLUSIONS: These results indicate that muscle tissue is a better source of stem cells than tendon tissue. Achievement of this study is proof that there is vast innate capacity of muscle tissue for enhancement of bone-tendon integration and ligamentization of ACL hamstring grafts and consequently muscle tissue should not be treated as waste after harvesting.

Quiescent Bone Lining Cells Are a Major Source of Osteoblasts During Adulthood.

The in vivo origin of bone-producing osteoblasts is not fully defined. Skeletal stem cells, a population of mesenchymal stem cells resident in the bone marrow compartment, are thought to act as osteoprogenitors during growth and adulthood. Quiescent bone lining cells (BLCs) have been suggested as a population capable of activation into mature osteoblasts. These cells were defined by location and their morphology and studies addressing their significance have been hampered by their inaccessibility, and lack of markers that would allow for their identification and tracing. Using lineage tracing models, we have observed labeled osteoblasts at time points extending beyond the reported lifespan for this cell type, suggesting continuous reactivation of BLCs. BLCs also make a major contribution to bone formation after osteoblast ablation, which includes the ability to proliferate. In contrast, mesenchymal progenitors labeled by Gremlin1 or alpha smooth muscle actin do not contribute to bone formation in this setting. BLC activation is inhibited by glucocorticoids, which represent a well-established cause of osteoporosis. BLCs express cell surface markers characteristic of mesenchymal stem/progenitors that are largely absent in osteoblasts including Sca1 and Leptin Receptor. BLCs also show different gene expression profiles to osteoblasts, including elevated expression of Mmp13, and osteoclast regulators RANKL and macrophage colony stimulating factor, and retain osteogenic potential upon transplantation. Our findings provide evidence that bone lining cells represent a major source of osteoblasts during adulthood. Stem Cells 2016;34:2930-2942.

Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation.

AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs). METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers - alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry. RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein. CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage.

Effect of UVC radiation on mouse fibroblasts deficient for FAS-associated protein with death domain.

PURPOSE: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. MATERIALS AND METHODS: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. RESULTS: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. CONCLUSIONS: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.

Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.

Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo.

Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model.

PURPOSE: Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. METHODS: Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. RESULTS: We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. CONCLUSION: We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

Preosteocytes/osteocytes have the potential to dedifferentiate becoming a source of osteoblasts.

Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while the negative population showed evidence of adipogenesis. We observed numerous Dmp1Cre-labeled osteoblasts on the surface of bone chips following their in vivo transplantation. Our data indicate that cells embedded in bone matrix are motile, and once given access to the extra bony milieu will migrate out of their lacunae. This population of cells is phenotypically and functionally heterogeneous in vitro. Once the preosteocytes/osteocytes leave lacunae, they can dedifferentiate, potentially providing an additional source of functional osteoblasts.

The effect of two different bracket types on the salivary levels of S mutans and S sobrinus in the early phase of orthodontic treatment.

OBJECTIVE: To determine the difference in the levels of Streptococcus mutans and S sobrinus in stimulated saliva in orthodontic patients with different bracket types (stainless steel and esthetic brackets) using polymerase chain reaction and cultivation method. MATERIALS AND METHODS: Thirty-two patients, aged 13 to 30 years, were selected following these criteria: 1) orthodontic treatment indication, 2) systemic health, and 3) no tobacco and antibiotic consummation for three months prior to the commencement of the study. Patients were divided into two groups according to the bracket type; 16 patients formed the conventional bracket group (stainless steel brackets), and 16 patients formed the esthetic bracket group (plastic brackets). The levels of S mutans and S sobrinus in stimulated whole saliva samples were collected prior to fixed orthodontic appliance placement (T1) and 12 weeks after placement (T2), as were the Decayed, Missing, and Filled Surface Index (DMFS) and Oral Hygiene Index-Simplified (OHI-S). Mann-Whitney, Wilcoxon, and chi-square tests were used for statistical analysis. RESULTS: Statistical analysis (chi-square test) showed no difference in S mutans and S sobrinus counts among patients with different brackets at either T1 or T2. There was no difference in total bacteria counts after fixed orthodontic appliance placement. CONCLUSION: The number of colony-forming units of S mutans and S sobrinus in stimulated saliva samples does not seem to be significantly different between patients with stainless steel brackets and patients with plastic brackets.

[Osteogenesis imperfecta and achievements in cell and gene therapy]. / Osteogenesis imperfecta i dostignuca u stanicnoj i genskoj terapiji.

Osteogenesis imperfecta (OI) or "brittle bone" disease is characterized by fragile bones, skeletal deformity, and growth retardation. Depending on the mutation and related phenotype, O1 is classified into types I-IV, which are caused by different mutations in collagen genes, and types V-VIII, which are indirectly but not directly collagen related. The most common cause of this inheritable disorder of connective tissue are mutations affecting the COL1A1 and COL1A2 genes of type I collagen. There is no cure for OI and current treatments include surgical intervention, use of prostheses and physical therapy. Pharmacological agents have also been tried with limited success, with the exception of recent use of bisphosphonates, which have been shown to have some effect in bone mass acquisition. Since OI is a genetic disease, these agents are not expected to alter the course of collagen mutations. Recent technology in molecular biology has led to the development of transgenic models of OI, which are necessary for development of cell and gene therapies as potential treatments for OI and are currently being actively investigated. However, the design of gene therapies for OI is complicated by genetic heterogeneity of the disease and by the fact that most of OI mutations are dominant negative where the mutant allele product interferes with the function of the normal allele. Therefore, therapy needs to include suppression of the mutant allele and introduction of the wild type allele. The present review will discuss the classification of OI and molecular changes seen in different types of OI and transgenic murine models that mimic different types of OI. Cell therapy, gene therapy, and a combination of both represent new approaches in OI therapy development that are being investigated as potential future treatments for OI. Modest success of cell therapy, encouraging results of gene therapy in vitro and in animal models as well as their problems and limitations for use in humans will be presented.