Multifaceted Characterization of Human Embryonic Stem Cell-Derived Mesenchymal Stem/Stromal Cells Revealed Amelioration of Acute Liver Injury in NOD-SCID Mice.

Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future.

A comparative study of the predictive value of four models for death in patients with severe burns.

OBJECTIVE: To assess the prognostic value of the Ryan score, Belgian Outcome of Burn Injury (BOBI) score,revised Baux (rBaux) score, and a new model (a Logit(P)-based scoring method created in 2020) for predicting mortality risk in patients with extremely severe burns and to conduct a comparative analysis. METHODS: A retrospective analysis was conducted on 599 burn patients who met the inclusion criteria and were admitted to the burn unit of the First Affiliated Hospital of Nanchang University from 2017 to 2022. Relevant information was collected, and receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA) were plotted for each of the four models in assessing mortality in these burn patients using both age-stratified and unstratified forms. The ROC curve section was further compared with the area under the curve (AUC), optimal cutoff value, as well as its sensitivity and specificity. Additionally, the quality of the AUC was assessed using the Delong test. RESULT: Among the patients who met the inclusion criteria, 532 were in the survival group and 67 in the death group. Irrespective of age stratification, the novel model exhibited superior performance with an AUC of 0.868 (95% CI: 0.838-0.894) among all four models predicting mortality risk in included patients, and also demonstrated better AUC quality than other models; the calibration curves showed that the accuracy of all four models was good; the DCA curves showed that the clinical utility of the novel model and rBuax score were better. In the comparison of four scoring models across different age groups, the new model demonstrated the largest AUC in both 0-19 years (0.954, 95% CI 0.914-0.979) and 20-59 years groups (0.838, 95% CI 0.793-0.877), while rBuax score exhibited the highest AUC in ≥ 60 years group (0.708, 95% CI of 0.602-0.800). The calibration curves showed that the four models exhibited greater accuracy within the age range of 20-59 years, while the DCA curves indicated that both the novel model and rBuax score scale displayed better prediction in both the 20-59 and ≥ 60 years groups. CONCLUSIONS: All four models demonstrate accurate and effective prognostication for patients with severe burns. Both the novel model and rBaux score exhibit enhanced prediction utility. In terms of the model itself alone, the new model is not simpler than, for example, the rBaux score, and whether it can be applied clinicallyinvolves further study.

Highly Adhesive Antibacterial Bioactive Composite Hydrogels With Controllable Flexibility and Swelling as Wound Dressing for Full-Thickness Skin Healing.

Polyzwitterionic hydrogels as skin wound dressings have been extensively studied owing to their superior antibacterial properties and skin adhesiveness, but their practical applications still suffer from a low adhesion strength and a high swelling ratio, which hinder the application of hydrogel for cutaneous healing. Here, we developed a novel biocompatible poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (PolySBMA) composite hydrogel with high stretchability, low swelling, strong skin adhesiveness, and antibacterial effect for enhancing wound healing. Naturally rigid polymers including quaternized chitosan methacrylate (QCSMA) and gelatin methacrylate (GelMA) are used as bioactive cross-linkers to endow PolySBMA/QCSMA/GelMA (SQG) hydrogel with a low swelling ratio and high bioactivity. The optimized hydrogel has excellent mechanical flexibility, with the ultimate tensile strength, tensile strain, modulus, and toughness of up to 344.5 kPa, 364%, 14.7 kPa, and 33.4 kJ m-3, respectively. The adhesiveness of the hydrogel to the skin tissue is as high as 38.2 kPa, which is critical for stopping the bleeding from the wound. The synergistic contributions from the PolySBMA and QCSMA endow hydrogel with good antibacterial properties against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Moreover, the natural polymer cross-linked polyzwitterionic hydrogel shows good cell activity, hemocompatibility, and histocompatibility. The in vivo full-thickness skin defect model demonstrates that the SQG hydrogel efficiently improves the granulation tissue formation and collagen deposition. In summary, such superiorly skin-adhesive antibacterial biocompatible hydrogel with controllable flexibility and swelling holds great promise as wound dressings for acute wounds.

Recurrence Is a Noticeable Cause of Rifampicin-Resistant Mycobacterium tuberculosis in the Elderly Population in Jiangxi, China.

Setting: Rifampicin-resistant tuberculosis (RR-TB) in elderly people in Jiangxi Province, China. Objective: To investigate the incidence of RR-TB and risk factors in elderly people within a hospital setting in China. Design: Retrospective cohort study. Methods: A comparative study was performed to analyze RR-TB and rifampicin-susceptible TB (RS-TB). The 15-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) method was used to distinguish between relapse and reinfection. Results: Twenty-three recurrent cases occurred in 151 elderly patients with RR-TB, and 24 recurrent cases occurred in 466 elderly patients with RS-TB during this period. TB recurrence was significantly different in the RR-TB and RS-TB groups (OR = 0.35, 95% CI: 0.14-0.88; χ2 = 5.28, P = 0.03). Comparing the risk factors for RR-TB and RS-TB, we found that educational level, age, and pulmonary cavity were inextricably linked to RR-TB in elderly patients. Furthermore, pulmonary cavity, HIV status, and alcohol consumption were associated with recurrence in elderly RR-TB patients. Conclusions: Recurrence is an important source of RR-TB in the elderly population. It is necessary to promptly treat tuberculosis patients, prevent the spread of AIDS, and reduce alcohol intake to control recurrent RR-TB in the elderly population.

Notch-1 inhibition reduces proliferation and promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

Low differentiation and high proliferation rates are critical factors affecting bone marrow mesenchymal stem cell (BMSC) tumorigenesis. The present study aimed to investigate the role of the Notch signaling pathway in BMSC proliferation and osteogenic differentiation. Mouse BMSCs were divided into control, vector, Notch1-small interfering (si)RNA, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups. The siRNA-Notch1, γ-secretase inhibitor, and Notch1-siRNA + γ-secretase inhibitor groups were treated with Notch1 siRNA and/or γ-secretase inhibitor. Following treatment, cell proliferation was evaluated using a Cell Counting Kit-8. Tumor-related factors, including transforming growth factor (TGF)-ß1, c-Myc and p53, were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. BMSC osteogenic differentiation was induced and the cells were stained with alizarin red at 14 and 21 days. Alkaline phosphatase (AKP) activity was also evaluated. The siRNA-Notch1 and γ-secretase inhibitor both reduced BMSC proliferation and the expression of TGF-ß1 and c-Myc and increased the expression of p53. Following the induction of osteogenesis and staining with alizarin red, the level of AKP was significantly higher in cells in the siRNA-Notch1 and γ-secretase inhibitor groups compared with that in the control group. It was found that Notch1 inhibition reduced proliferation and promoted the osteogenic differentiation of BMSCs.

[Integrative analysis of gene expression profile and DNA methylation profile of long-term cultivated porcine bone marrow mesenchymal stem cells].

Objective: To integrate the result of whole genome expression data and whole genome promoter CpG island methylation data, to screen the epigenetic modulated differentially expressed genes from transformed porcine bone marrow mesenchymal stem cells (BMSCs) after long-term cultivation. Methods: Bone marrow from 6 landrace pigs, 3-month-old about 50 kg weight, was aspirated from the medullary cavity of the proximal tibia. The BMSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The transfor mation of BMSCs was tested by several methods including cell morphology observation, karyotype analysis, clone forming in soft agarose, serum requirement assay, and tumor forming in mice. The Agilent Pig 4x44k Gene Expression Microarray was used to investigate the differentially expressed mRNA. The methylated genes expression profile was performed using customized pig methylation chip. The gene expression and DNA methylation profiles were integrated to find out the epigenetic modulated differentially expressed genes, and to complete the bioinformatic analysis. Results: BMSCs showed a change in appearance, from the initial spindle shape to a more flatted morphology then to small contact shape. After additional passages, BMSCs gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth, chromosomal abnormality, and tumor formation in nude mice. The gene chip analysis demonstrated that 257 genes were upregulated and 315 genes were downregulated during long-term cultures as well as multiple signal pathways transduction involved, such as cell cycle, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, pathways in cancer, and P53. The analysis from methylation chip of coding genes suggested epigenetic regulation was involved in BMSCs spontaneous transformation and play a important role on it; 962 genes were hypermethylated and 1219 genes were hypomethylated, which were involved in the biological process of cellular metabolic, structure, and tumor generation. The combined analysis of genes regulated by methylation in the transformation process of BMSCs found that the methylation changes of the 35 genes were contrary to the direction of expression change (correlation coefficient r=-0.686, P=0.000); in which the methylation level of 21 genes promoter regions were increased while the gene expression decreased, and the methylation level of the 14 genes promoter regions decreased and the gene expression increased. At the same time, KEGG enrichment analysis revealed multiple genes regulated by methylation, involved in stem cell differentiation and multiple cell signaling pathways. Among the 14 down-regulated genes, many of them have the role of regulating the interaction of tumor and immunization, and the change of the methylation status of the CDKN3 promoter region may be closely related to the cell oncology. Conclusion: The results deepen our understanding of the crucial role of coding genes methylation modification in BMSCs transformation, and may provide new approach to establish safe criteria for BMSCs clinical applications and transformation prevention.

Biological function evaluation and effects of laser micro-pore burn-denatured acellular dermal matrix.

OBJECTIVE: In the field of burns repairs, many problems exist in the shortage of donor skin, the expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity function after wound healing. Previous studies showed that burn-denatured skin could return to normal dermis formation and function. This study investigates the application of laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in the repair of burn wounds and evaluates the biological properties and wound repair effects of DADM in implantation experiments in Kunming mice. METHODS: Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II° burn wound was created on the dorsum of the mice by an electric heated water bath. The full-thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels and subcutaneous implantation experiments in Kunming mice. RESULTS: We found statistical significance (P<0.05) of the elastic modulus (MPa), maximum load force (N) and contraction measurement (CM) of the laser micro-pore burn-DADM (experimental group) compared to the control group (no laser micro-pore burn-DADM). Cytokine expression level was different in the dermal matrixes harvested at various time points after burn (24h, 48h, 72h and infected wound group). Comparing the dermal matrix from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the dermal matrix were observed in both experimental and control groups (24h burn group), while the obviously vascular infiltration and fiber fusion were observed in the experimental group after subcutaneous implantation experiments. CONCLUSION: There was better bio-performance, low immunogenicity and better dermal incorporation after treated by laser micro-pore drilling and decellularized deep II° burn-DADM, which may be considered as a better substitute for dermal matrix. Furthermore, the earlier harvested DADM after burn (24h) shows the better transplantation effect.

Methylation-mediated silencing of miR-124 facilitates chondrogenesis by targeting NFATc1 under hypoxic conditions.

Chondrogenic differentiation of mesenchymal stem cells is regulated by many different pathways. Recent studies have established that hypoxia and epigenetic alterations potently affect expression of chondrogenesis marker genes. Sox9 is generally regarded as a master regulator of chondrogenesis and microRNA-124 (miRNA-124) regulates gene expression in murine bone marrow-derived mesenchymal stem cells. Therefore, in this study we investigated whether epigenetic regulation of miRNA-124 could affect the expression of Sox9 and thereby regulate chondrogenesis. A cell pellet culture model was used to induce chondrogenesis in C3H10T1/2 cells under hypoxic conditions (2% O2) to determine the effects of hypoxia on miR-124 expression and DNA methylation. The expression of miR-124 was significantly downregulated under hypoxic conditions compared to normoxic conditions (21% O2). The expression of chondrogenesis marker genes was significantly increased under hypoxic conditions. Bisulfite sequencing of the CpG islands in the promoter region of miR-124-3 showed that CpG methylation was significantly increased under hypoxic conditions. Treating the cells with the DNA demethylating agent 5'-AZA significantly increased miR-124 expression and decreased expression of markers of chondrogenesis. Overexpressing miR-124 under hypoxic conditions inhibited NFATc1 reporter activity. NFATc1 was shown to bind to the promoter region of Sox9. Taken together, our data provide evidence that miR-124 acts as an inhibitor of NFATc1. Under hypoxic conditions when miR-124 is downregulated by methylation of CpG islands in the promoter, NFATc1 can bind to the Sox9 promoter and induce the expression of Sox9 leading to chondrogenesis. These results support the role of epigenetic regulation in establishing and maintaining a chondrogenic phenotype.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds.

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

An overview on bone protein extract as the new generation of demineralized bone matrix.

Bone protein extract is regarded as the new generation of demineralized bone matrix. The aim of this paper is to describe and characterize the properties of demineralized bone matrix and its new generation product in addition to its application in animal and human studies. Bone protein extract has features of osteoconductivity, osteoinductivity and osteogenicity, which originate from its unique and precise processing. It has exhibited powerful bone formation capacity both in animal experiments and in clinical trials by providing an optimal microenvironment for osteogenesis. Furthermore, not only does it have excellent biocompatibility, it also has good compatibility with other implant materials, helping it bridge the host and implanted materials. Bone protein extract could be a promising alternative for demineralized bone matrix as a bone graft substitute.

T1ρ-MR imaging technique to distinguish four-layered zonal structure of articular cartilage for detecting early-onset osteoarthritis.

In order to evaluate the capacity of T1ρ-MRI(Magnetic Resonance Imaging) technique in distinguishing a four-layered zonal structure of articular cartilage for detecting the early-onset osteoarthritis (OA), eight 3-month-old female Danish landrace pigs, weighing 45.3 ± 5.5 kg, were used in this study. Two hind knees were randomly divided into two groups. IL-1ß was injected into articular cavities in one group while physiological saline into another as a control one. Six hours later, the patellae were obtained for T1ρ-MRI and histological examination to distinguish the four-layered zonal structure of articular cartilages. The thickness of each layer and their percentages in the entire cartilage were measured on both T1ρ-MR images and the histological slices. T1ρ values of every layer were calculated from T1ρ-mapping images. In the result, the four-layered zonal structure of articular cartilage was displayed on both T1ρ-MR images and the histological slices. The ICC between these two methods was 0.981 for single measures (P=0.001), and 0.990 for average measures (P = 0.001). Compared with control patellae, T1ρ values significantly increased on the superficial and calcified layers in the IL-1ß treated patellae (P=0.037). Therefore, this study demonstrates that the four-layered zonal structure of the articular cartilage is visibly displayed on the T1ρ-MR images. T1ρ-MRI technique can detect the earliest event of cartilage degeneration while these measurable changes are not shown in the histological slices. It is possible to use this technique for observing the effects of early interventions on the early-onset OA.

A traditional Chinese medicine formula extracts stimulate proliferation and inhibit mineralization of human mesenchymal stem cells in vitro.

AIM OF THE STUDY: To investigate the effects of a traditional Chinese medicine (TCM) formula extract, named as ZD-I, on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. MATERIALS AND METHODS: When hMSCs cultivated in the basal medium with ZD-I, cell viability was assessed by MTT assay and cellular proliferation was assessed by SYBR green I assay. The effects of ZD-I on osteogenic differentiation of hMSCs were assessed by alkaline phosphatase (ALP) activity, mineralization assay and real-time RT-PCR. RESULTS: ZD-I (0.78-100 microg/ml) was non-cytotoxic. The 50% inhibitory concentration (IC50) of hMSCs was 200 microg/ml. ZD-I (0.78-50 microg/ml) stimulated the proliferation of hMSCs. ZD-I did not change ALP activity of hMSCs cultivated in osteogenic medium in the early stage (4 and 7 days), but ZD-I inhibited the mineralization of hMSCs through down-regulation of several osteogenic markers (e.g. osteocalcin, bone morphogenetic protein 2 and osteopontin) in the late stage. CONCLUSIONS: ZD-I stimulate cellular proliferation and decrease the bone mineral deposition of hMSCs. These results suggest ZD-I may play an important therapeutic role in osteoarthritic patients by improving proliferative capacity of hMSCs and inhibiting the mineralization of hMSCs.

Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion.

To elucidate the molecular mechanisms of spinal fusion with different graft materials during an anterior lumbar interbody fusion, we examined the gene-expression profiles after implantation of equine bone protein extract, rhBMP-2 and autograft using microarray technology and data analysis, including hierarchical clustering, self-organizing maps (SOM), KEGG pathway and Biological process GO analyses in a porcine model. The results suggest that equine bone protein extract exhibited a more similar expression pattern with autograft than that of rhBMP-2. rhBMP-2 recruits progenitor cells, proliferation and differentiation possibly by inducing various factors including PGHS-2, IFGBP-2, VEGF and chemokines and then leads to preferable membranous ossification and bone remodeling. Conversely, equine bone protein extract results in endochondral ossification via upregulation of cartilage-related genes. Ossification by inducing direct osteoblastic differentiation and obviating the cartilaginous intermediate phases may increase spinal fusion rate.

ISSLS prize winner: positron emission tomography and magnetic resonance imaging for monitoring interbody fusion with equine bone protein extract, recombinant human bone morphogenetic protein-2, and autograft.

STUDY DESIGN: Prospective and randomized experimental study with anterior lumbar interbody fusion in a porcine model. OBJECTIVE: To assess the early time-course of spinal fusion with equine bone protein extract (COLLOSS E), recombinant human bone morphogenetic protein-2 (rhBMP-2), and autograft using quantitative methods of positron emission tomography (PET)/computed tomography and magnetic resonance imaging (MRI). SUMMARY OF BACKGROUND DATA: Different growth and differentiation factors are currently being used for inducing bone formation in spinal fusion. However, the mechanisms and time-course of bone formation using these graft substitutes remain less known. METHODS: Eighteen female Danish landrace pigs underwent a 3-level anterior lumbar interbody fusion procedure from L3-L6. A PEEK cage, packed with COLLOSS E, rhBMP-2, or autograft, was randomly placed. Each group of 6 pigs was observed for 2, 4, or 8 weeks, respectively. F PET/computed tomography and MRI examinations were performed, and data were correlated with histomorphometry. PET data were analyzed using a Gjedde-Patlak plot. K-values from the plot correspond to the metabolic rate. T2-values were calculated by T2 mapping. RESULTS: rhBMP-2 presented the highest bone formation on histologic sections at 25.6% at 4 weeks after surgery. Eight weeks after surgery, autograft had the highest bone formation with 37.3%, which was significantly higher than rhBMP-2 at 30.5% (P < 0.05), and higher than COLLOSS E at 27.0% (P = 0.06). COLLOSS E and rhBMP-2 had significantly higher K-values than autograft (P < 0.05) at 2 weeks after surgery. There were no differences in K-values between COLLOSS E and autograft at 4 and 8 weeks. However, rhBMP-2 was significantly higher at 4 weeks and lower at 8 weeks than these 2 (P < 0.05). Linear correlation, R = 0.8275, was observed for intertrabecular volume/total volume and T2-values. CONCLUSION: PET and MRI are valid tools for monitoring the process of interbody fusion in vivo. Osteogenic mechanisms using COLLOSS E resembles that of autograft by the process of endochondral ossification. rhBMP-2 deposits osteoid directly on the collagen network.

Molecular treatment strategies and surgical reconstruction for metastatic bone diseases.

Molecular mechanism of bone metastasis development is extremely complex. It is determined by intrinsic properties of cancer cells or cancer stem cells (CSCs) and intricate bone microenvironment. Therefore, molecular treatment strategies have been suggested to directly induce cancer cells apoptosis and to target vascular and bone microenvironment as well, thus inhibiting vicious cycles established between osteoblasts/osteoclasts and metastatic cancer cells. Chemokine/chemokine receptor pathway, adhesion molecules, and proteinases are crucial for bone metastatic process, including migration, adhesion and invasion into bone, angiogenesis and cell proliferation, which could provide potential targets for prevention and treatment of bone metastasis. Restoration of metastasis suppressor genes and microRNAs inhibits bone metastasis. Furthermore, targeting the bone marrow endothelium around cancer cells by use of both antiangiogenic inhibitors and vascular disrupting agents is another promising and valid therapeutic approach. On the other hand, many antitumor drugs/small molecules are limited in reaching tumor site due to a very complex vasculature. Nanotechnology aids in the targeted delivery of antitumor drugs/small molecules. For severe bone lesion, multifunctional implants integrating with antitumor drugs/small molecules and bone forming factors could be effective to reconstruct bone defects and to improve the quality of life in patients with bone metastasis.

Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro.

Hyaluronan (HA) plays a predominant role in tissue morphogenesis, cell migration, proliferation, and cell differentiation. The aims of the present study were to investigate whether (i) prolonged presence of high concentration (4.0 mg/mL) 800 KDa HA and (ii) pretreatment with HA can modify osteogenic differentiation of pig bone marrow stromal cells (pBMSC). Cell proliferation and mineralization were measured. Expression of differentiation-related genes was evaluated by means of real-time reverse transcription polymerase chain reaction (RT-PCR). HA increased cell proliferation on day 7. HA decreased the basal level of bone-related gene expression and increased the basal level of sox9 marginally during 7-day pretreatment with HA. HA increased calcium deposit on day 21. cbfa1, ALP, and type 1 alpha collagen (Col1) expression was increased when pBMSC were cultivated in osteogenic medium, whereas their expression was decreased in the presence of HA on day 7. On day 14, the addition of HA upregulated cbfa1 and ALP expression compared to osteogenic medium group; there was no significant difference in Col1 expression. At day 21, osteocalcin (OC) expression showed 2.5-fold upregulation over osteogenic medium. These results suggest that exogenous HA stimulates endogenous HA, which together may play a synergetic role in osteogenic differentiation under osteoinducing conditions although gene expression was inhibited at the early stage.

Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern.

There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker genes. However, it is not fully clear whether multilineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) of BMSC is associated with a specific gene expression pattern. In the present study, we investigated the gene expression pattern of representative transcription factors and marker genes along those three mesenchymal lineages during a particular lineage differentiation of porcine BMSC by means of real-time PCR measurement. In an osteogenic medium, the mRNA levels of cbfa1, osterix, alkaline phosphatase, type 1 collagen, osteonectin, bone sialoprotein, and osteocalcin were induced stepwise. Meanwhile, sox9, specific to chondrogenic differentiation, was inhibited but not PPARgamma2 specific to adipogenic differentiation. In an adipogenic medium, adipogenic differentiation was confirmed by upregulation of PPARgamma2 and aP2 and downregulation of osteogenic genes and sox9. Chondrogenic differentiation was induced in cell pellet culture by expression of sox9, type 2 collagen, and aggrecan. Cbfa1 and PPARgamma2 were inhibited in chondrogenic medium. These results indicate that the differentiation potential of BMSC to a particular mesenchymal lineage relies upon specific gene expression pattern, namely upregulation of genes specific for this lineage and suppression of other lineage differentiation.