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Apart from aging process, adult intervertebral disc (IVD) undergoes various degenerative processes. However, the nicotine has not been well identified as a contributing etiology. According to a few studies, nicotine ingestion through smoking, air or clothing may significantly accumulate in active as well as passive smokers. Since nicotine has been demonstrated to adversely impact various physiological processes, such as sympathetic nervous system, leading to impaired vasculature and cellular apoptosis, we aimed to investigate whether nicotine could induce IVD degeneration. In particular, we evaluated dose-dependent impact of nicotine in vitro to simulate its chronic accumulation, which was later treated by platelet-derived biomaterials (PDB). Further, during in vivo studies, mice were subcutaneously administered with nicotine to examine IVD-associated pathologic changes. The results revealed that nicotine could significantly reduce chondrocytes and chondrogenic indicators (Sox, Col II and aggrecan). Mice with nicotine treatment also exhibited malformed IVD structure with decreased Col II as well as proteoglycans, which was significantly increased after PDB administration for 4 weeks. Mechanistically, PDB significantly restored the levels of IGF-1 signaling proteins, particularly pIGF-1 R, pAKT, and IRS-1, modulating ECM synthesis by chondrocytes. Conclusively, the PDB impart reparative and tissue regenerative processes by inhibiting nicotine-initiated IVD degeneration, through regulating IGF-1/AKT/IRS-1 signaling axis.

Background Recurrent aphthous stomatitis (RAS) is considered a prophase symptom in patients with specific cancers. This study assessed the association between RAS and subsequent onset of cancer based on a nationwide population‐based database in Taiwan. Materials and Methods We selected study participants from the National Health Insurance Research Database from January 2000 to December 2008. Patients in the non‐RAS cohort were matched to case study patients at a 1:1 ratio through frequency matching. All participants were followed up for at least 5 years, and those who received cancer diagnoses during follow‐up were identified. Results Among 52 307 patients with and 52 304 patients without RAS, the combined hazard ratio (HR) of all subsequent cancer cases was 1.3 (95% confidence interval [CI]: 1.25‐1.35, P = 0). RAS diagnosis was associated with risk for cancers of the head and neck (aHR = 2, 95% CI: 1.8‐2.3), colon (aHR = 1.2, 95% CI: 1.1‐1.4), liver (aHR = 1.1, 95% CI: 1‐1.3), pancreas (aHR = 1.4, 95% CI: 1.1‐1.7), skin (aHR = 1.4, 95% CI: 1.2‐1.7), breast (aHR = 1.2, 95% CI: 1.1‐1.4), and prostate (aHR = 1.5, 95% CI: 1.3‐1.8), as well as hematologic cancers (aHR = 1.6, 95% CI: 1.3‐1.9). A higher risk was observed for male patients (aHR = 1.35, 95% CI: 1.28‐1.42) than for female patients (aHR = 1.25, 95% CI: 1.18‐1.31) with RAS. Conclusions RAS was associated with specific cancers. Susceptible RAS patients should be screened for specific cancers.

Complications of diabetes mellitus (DM) range from acute to chronic conditions, leading to multiorgan disorders such as nephropathy, retinopathy, and neuropathy. However, little is known about the influence of DM on intervertebral disc degeneration (IVDD). Moreover, traditional surgical outcomes in DM patients have been found poor, and to date, no definitive alternative treatment exists for DM-induced IVDD. Recently, among various novel approaches in regenerative medicine, the concentrated platelet-derived biomaterials (PDB), which is comprised of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), etc., have been reported as safe, biocompatible, and efficacious alternatives for various disorders. Therefore, we initially investigated the correlations between DM and IVDD, through establishing in vitro and in vivo DM models, and further evaluated the therapeutic effects of PDB in this comorbid pathology. In vitro model was established by culturing immortalized human nucleus pulposus cells (ihNPs) in high-glucose medium, whereas in vivo DM model was developed by administering streptozotocin, nicotinamide and high-fat diet to the mice. Our results revealed that DM deteriorates both ihNPs and IVD tissues, by elevating reactive oxygen species (ROS)-induced oxidative stress, inhibiting chondrogenic markers and disc height. Contrarily, PDB ameliorated IVDD by restoring cellular growth, chondrogenic markers and disc height, possibly through suppressing ROS levels. These data imply that PDB may serve as a potential chondroprotective and chondroregenerative candidate for DM-induced IVDD.

// Bou-Yue Peng 1 , Chi-Sheng Chiou 2, 3 , Navneet Kumar Dubey 4, 5 , Sung-Hsun Yu 4, 6 , Yue-Hua Deng 4, 7 , Feng-Chou Tsai 8 , Han-Sun Chiang 7 , Ying-Hua Shieh 10, 11 , Wei-Hong Chen 4 and Win-Ping Deng 2, 4, 9 1 Oral and Maxillofacial Surgery Section, Department of Dentistry, Taipei Medical University Hospital, Taipei, Taiwan 2 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 3 Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan 4 Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 5 Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan 6 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 7 Department of Life Science, Fu Jen Catholic University, Taipei, Taiwan 8 Department of Stem Cell Research, Cosmetic Clinic Group, Taipei, Taiwan 9 Graduate Institute of Basic Medicine, Fu Jen Catholic University, Taipei, Taiwan 10 Department of Family Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 11 Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan Correspondence to: Win-Ping Deng, email: wpdeng@tmu.edu.tw Keywords: osteoarthritis (OA), immortalized bone marrow stem cells (iBMSCs), cartilage regeneration, human papilloma virus (HPV)-16 E6/E7, molecular imaging Received: July 14, 2017 Accepted: August 27, 2017 Published: September 27, 2017 ABSTRACT Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.

Though the cross-induction of either acute kidney (AKI) injury to ischemic stroke (IS) or IS to AKI might not be encountered in the early stages of cerebrorenal syndrome (CRS), both pathologies coexist in late stages. Therefore, we firstly established a late stage CRS rat model by simultaneous induction of both diseases, and further, cerebro and reno-protective activities of human platelet-rich plasma (hPRP), a blood-derived tissue engineering biomaterial, were tested in this pathology. hPRP was administrated via left common carotid artery and abdominal aorta 2 h post-sham procedure in Sprague-Dawley rats. Circulatory inflammatory markers (TNF-α/MPO/IL-6/Ly6G/CD11b/c), histopathologic cerebro and renal changes and oxidative stress were determined. Inflammation, infarct size, brain-associated inflammatory/DNA and mitochondrial damage and oxidative-stress with reduced neurons and neurological function were manifested in CRS group compared to other groups. CRS group also demonstrated declined renal function, accelerated renal collagen deposition, fibrosis and compromised glomerular podocyte components (podocin/ZO-1/fibronectin/synaptopodin). However, hPRP simultaneously suppressed all the inflammatory, cerebral and renal pathologic characteristics. hPRP also inhibited the expression of brain-associated inflammatory/DNA/mitochondrial damage and oxidative-stress biomarkers. These findings imply that hPRP may effectively exert cerebro- and renoprotective activities in late stage CRS through anti-oxidative, anti-inflammatory, anti-DNA and anti-mitochochondrial damaging activities.

Tissue engineering is a burgeoning research field of regenerative medicine that holds promise to restore tissues and organs affected by traumatic injury, chronic disorders, age-induced degeneration, and other factors. Specifically, it consists of replacing or repairing damaged or diseased tissues by using natural, synthetic, or semisynthetic tissue mimics that could either be fully functional or will grow into required functionality. Recent progresses in cartilage tissue engineering represents a promising approach for regenerating articular cartilage through tissue-engineered scaffolds in vitro for subsequent implantation in vivo for the treatment of osteoarthritis. Being avascular, aneural, and alymphatic, the articular cartilage possesses limited capacity to intrinsic repair after damage from injury or degenerative disease. Therefore, current research is being directed to achieve a complete regeneration and repair. In this chapter, we have addressed the readers with an overview of structural component of knee joint and various bioinstructive polymeric hydrogel scaffolds for regeneration of cartilage.

Knee osteoarthritis (OA) is a chronic degenerative disorder which could be distinguished by erosion of articular cartilage, pain, stiffness, and crepitus. Not only aging-associated alterations but also the metabolic factors such as hyperglycemia, dyslipidemia, and obesity affect articular tissues and may initiate or exacerbate the OA. The poor self-healing ability of articular cartilage due to limited regeneration in chondrocytes further adversely affects the osteoarthritic microenvironment. Traditional and current surgical treatment procedures for OA are limited and incapable to reverse the damage of articular cartilage. To overcome these limitations, cell-based therapies are currently being employed to repair and regenerate the structure and function of articular tissues. These therapies not only depend upon source and type of stem cells but also on environmental conditions, growth factors, and chemical and mechanical stimuli. Recently, the pluripotent and various multipotent mesenchymal stem cells have been employed for OA therapy, due to their differentiation potential towards chondrogenic lineage. Additionally, the stem cells have also been supplemented with growth factors to achieve higher healing response in osteoarthritic cartilage. In this review, we summarized the current status of stem cell therapies in OA pathophysiology and also highlighted the potential areas of further research needed in regenerative medicine.

Osteoarthritis (OA) is a common disease associated with tissue inflammation, physical disability and imbalanced homeostasis in cartilage. For advanced treatments, biological approaches are currently focused on tissue regeneration and anti-inflammation. This study was undertaken to evaluate the therapeutic efficacies of hyaluronic acid (HA) and platelet-rich plasma (PRP) (HA+PRP) on OA. Articular chondrocytes were obtained from five OA patients. The optimal HA and PRP concentrations were evaluated by MTT assay. The expressions of chondrogenic and inflammatory genes were analyzed by RT-PCR. Signaling pathway was examined by immunoblotting and the expressions of OA pathology-related chemokines and cytokines was demonstrated by real-time PCR-based SuperArray. The therapeutic efficacies of HA+PRP were then demonstrated in 3D arthritic neo-cartilage and ACLT-OA model. Here we showed that HA+PRP could greatly retrieve pro-inflammatory cytokines-reduced articular chondrocytes proliferation and chondrogenic phenotypes, the mechanism of which involve the sequential activation of specific receptors CD44 and TGF-βRII, downstream mediators Smad2/3 and Erk1/2, and the chondrogenic transcription factor SOX9. The real-time PCR-based SuperArray results also indicated that OA pathology-related chemokines and cytokines could be efficiently suppressed by HA+PRP. Moreover, the cartilaginous ECM could be retrieved from inflammation-induced degradation by HA+PRP in both 2D monolayer and 3D neo-cartilage model. Finally, the intra-articular injection of HA+PRP could strongly rescue the meniscus tear and cartilage breakdown and then decrease OA-related immune cells. The combination of HA+PRP can synergistically promote cartilage regeneration and inhibit OA inflammation. This study might offer an advanced and alternative OA treatment based on detailed regenerative mechanisms.

Knee osteoarthritis (OA) is one of the most prevalent disorders in elderly population. Among various therapeutic alternatives, we employed stromal vascular fraction (SVF), a heterogeneous cell population, to regenerate damaged knee cartilage. OA patients were classified on the basis of age, gender, body mass index (BMI), and x-ray-derived Kellgren&ndash, Lawrence (KL) grade. They were treated with SVF and followed-up for 24 months. Visual analogue scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index were used to determine treatment efficacy. Cartilage healing was assessed using the MRI-based Outerbridge score (OS) and evaluation of bone marrow edema (BME) lesions, while a placebo group was used as a control. Time- and KL-dependent changes were also monitored. We observed a decreasing trend in VAS score and WOMAC index in the SVF-treated group up to 24 months, as compared with the placebo group. Besides, a significant increase and decrease in Lysholm and OS, respectively, were observed in the treatment group. Compared with the values before treatment, the greatly reduced WOMAC scores of KL3 than KL2 groups at 24 months, indicate more improvement in the KL3 group. Highly decreased BME in the treated group was also noted. In conclusion, the SVF therapy is effective in the recovery of OA patients of KL3 grade in 24 months.

Background: Recent studies have shown evidence that higher adiposity in the infrapatellar fat pad (IFP) induces inflammatory phenotypes in the knee joint and thereby contributes to the development and progression of osteoarthritis (OA). In particular, IFP adipocyte–derived inflammatory cytokines participate in pathological events. Our previous research has already addressed the therapeutic efficacy of hyaluronic acid and platelet-rich plasma (HA+PRP), including the promotion of cartilage regeneration and the inhibition of inflammation. The current study aimed to explore the remedial action of coadministered HA+PRP in OA recovery via IFP adipocyte inhibition. Hypothesis: HA+PRP repairs OA articular cartilage by inhibiting the release of adipokines from IFP adipocytes. Study Design: Controlled laboratory study. Methods: IFP adipocytes and articular chondrocytes were obtained from 10 patients with OA, and the effects of releasates containing cytokines and adipokines in IFP adipocyte–derived conditioned medium (IACM) on articular chondrocytes and IFP adipocytes themselves were evaluated. The therapeutic efficacy of exogenous HA+PRP was determined through its administration to cocultured IFP adipocytes and articular chondrocytes and further demonstrated in a 3-dimensional (3D) arthritic neocartilage model. Results: The IACM and IFP adipocyte–induced microenvironment could induce dedifferentiated and inflammatory phenotypes in articular chondrocytes. HA+PRP decreased the inflammatory potential of IFP adipocytes through the profound inhibition of cytokines and adipokines. The IACM-mediated and -reduced cartilaginous extracellular matrix could also be recovered through HA+PRP in the 3D arthritic neocartilage model. Conclusion: IFP adipocyte–derived releasates mediated inflammatory response dedifferentiation in chondrocytes, which was recovered through HA+PRP administration. Clinical Relevance: Our findings demonstrated that HA+PRP effectively diminished IFP adipocyte–promoted inflammation in articular chondrocytes, indicating that the IFP could be a potential therapeutic target for OA therapy.

Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age50) and OhNP (from old donor, age50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.

Adipose-derived stem cells (ADSCs) have been shown to be pluoripotent and explored for their usage in tissue engineering. Previously, we have established a cell-based approach comprised of platelet-enriched plasma and osteo-progenitor cells for treating osteoporosis in an ovariectomized-senescence-accelerated mice (OVX-SAMP8) model. In the present study, we intend to explore the feasibility of using ADSCs as a cell-based therapeutic approach for treating osteoporosis, and to examine the effects of aging on the pluoripotency of ADSCs and the efficiency of bone formation both in vitro and in vivo. Flow cytometry was used to characterize ADSCs isolated from young and aged female SAMP8 mice and showed that the highly positive expression of surface markers such as CD44 and CD105 and negative for CD34 and CD45. Therefore, to compare the aging effects on the growth kinetics and differentiation potential of young and aged ADSCs, we found that there was a significant decline in both the proliferation rate (approximately 13.3%) and osteo-differentiation potential in aged ADSC. Subsequently, young and aged ADSCs were transplanted into the bone marrow of osteoporotic mice (OVX-SAMP8) to evaluate their bone formation ability. ADSC transplants were shown effective in restoring bone mineral density in the right/left knees, femurs and spine, 4 months post-transplantation; mice which received young ADSC transplants showed significantly higher bone regeneration (an average of 24.3% of improved BMD) over those received aged ADSCs. In conclusion, these findings showed that aging impedes osteoporosis-ameliorating potential of ADSC by diminishing osteogenic signal, and that ADSC could be used as a potential cell-based therapy for osteoporosis.

This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1β (10 ng/ml)+TNF-α (20 ng/ml). First, the MTT result indicated that 1 ng/ml TGF-β1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1β+TNF-α, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1β+TNF-α were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin α1β1 and CD44 were strongly inhibited by IL-1β+TNF-α, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1β+TNF-α. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.

Polyethyleneimine (PEI) is a cationic polymer that is effective in gene delivery in vivo. Plasmid DNA incorporating the Sleeping-Beauty (SB) transposon has been shown to induce long-term transgene expression in mouse lungs after PEI-mediated delivery. In the current report, we followed the reporter gene expression mediated by PEI/SB delivery in lungs of mice using the non-invasive bioluminescent imaging (BLI) technology. After delivery, the reporter gene signal showed a rapid decay in the first two weeks to a nearly undetectable level, but then the signal augmented gradually in the following weeks and finally reached a stable level that maintained until the natural death of animals. The stabilization of transgene expression is associated with the multiplication of a small number of PEI/SB-labeled alveolar cells, which proliferated both under normal conditions and in response to acute local injury for epithelia repair, and may play a role in long-term homeostatic maintenance in alveoli. The data presented here suggests that systemic delivery of PEI/SB induces stable transfection specifically in a small population of alveolar progenitor cells. The technique provides a promising platform for future research in distal lung biology and tissue regenerative therapy.

Colorectal glands are important functional organs in colorectal tissue and are also the origin of colorectal carcinomas. Epithelial cell polarization of colorectal glands is related to structural integrity and physiological functions of colorectal glands as well as colorectal carcinoma formation. The cellular apoptosis susceptibility (CSE1L/CAS) protein has been shown to induce polarity formation of human colorectal cells in cell culture. E-cadherin expression in epithelial cells is crucial for the establishment and maintenance of epithelial cell polarity. In this study we examined the distributions of CSE1L and E-cadherin in the epithelial glands of normal and neoplastic colorectal epithelium and correlated these to polarity formation in the colorectal glands. Our results showed that CSE1L was differentially stained in the epithelial glands of neoplastic colorectal epithelium, and the staining was related to gland epithelial cell polarization and E-cadherin distribution. CSE1L was associated E-cadherin in GST pull-down experiments and immunoprecipitation assays. Basolateral staining of CSE1L and E-cadherin were seen in the polarized glands of normal and neoplastic colorectal epithelium. Absence of basolateral CSE1L staining in neoplastic epithelium glands was associated with loss of gland epithelial cell polarity, and this was parallel with E-cadherin staining. The non-polarized areas in epithelium glands showed a patchy staining for CSE1L and E-cadherin. These results indicate that examination of CSE1L and E-cadherin distribution in colorectal epithelium glands may be valuable for evaluating the malignance of colorectal disease.

The aim of this study was to develop a cell-based bone-regeneration approach evaluated by molecular imaging and immunohistochemistry. Methods: Genetically modified NIH3T3 embryonic fibroblasts carrying enhanced green fluorescent protein (NIH3T3-G) were predifferentiated into osteoblastlike cells using platelet-rich plasma (PRP) medium, followed by intraosseous transplantation into ovariectomized senescence-accelerated mouse prone substrain 8 (OVX-SAMP8 mice). Results: PRP-conditioned NIH3T3-G (PRP/NIH3T3-G) engraftment prevented the development of osteoporosis. Molecular imaging and immunohistochemistry demonstrated the migration of NIH3T3-G cells from the implantation site throughout the skeleton. In situ analyses revealed coexpression of osteopontin and green fluorescent protein in the newly formed bone tissue, demonstrating that the transplant restored the bone trabecular architecture and mineral density in treated OVX-SAMP8 mice. Interestingly, the life span of OVX-SAMP8 mice receiving PRP/NIH3T3-G transplantation was significantly prolonged and similar to that of the congenic senescence-resistant strain of mice. Conclusion: This unique and yet simple approach could potentially be applied to the treatment of senile postmenopausal osteoporosis and perhaps inborn genetic syndromes associated with accelerated aging, such as Hutchinson–Gilford progeria syndrome, and for the prolongation of life expectancy in general.

Although pressure therapy is the mainstay of treatment for hypertrophic scars, its actual mechanism remains unknown. An in vitro study was designed to investigate the effects of positive pressure on the growth of human scar-derived fibroblasts through its transforming growth factor beta1 (TGF-beta1) secretion. A pneumatic pressure system connecting to a cell culture chamber was designed. Six-well cultured plates with fibroblasts implanted were treated with different pressure settings. Cells were treated with constant pressure 20 mm Hg above atmosphere pressure (group A n = 18) or with 40 mm Hg above atmosphere pressure (group B n = 18) daily for nine successive days. Cells without pressure were treated as the control study (group C n = 6). Each experimental group was divided into daily pressure applied at 24 hours (n = 6), 18 hours (n = 6), and 12 hours (n = 6). Cell counting was performed on the 2nd, 4th, 7th, 9th, 11th, and 14th day after implantation. On day 4, the concentration of transforming growth factor beta1 was measured, and cell doubling time was calculated. Compared with the control group, there was a significant decrease in cell count and the concentration in the 18-hour and 24-hour 20 mm Hg or 40 mm Hg pressure treated group. The cell doubling time was significantly increased in the 24-hour 20 mm Hg or 40 mm Hg pressure treated groups, and the 18-hour 40 mm Hg pressure treated group. (P < .05) Pressure inhibits the growth and activity of human scar fibroblasts, and a higher pressure application can shorten the daily application period. There should be an optimal pressure level corresponding to a daily application period to achieve the most effective results on pressure therapy for scars.

Microtia is characterized as an incomplete auricular development and surgical reconstruction for microtia is still limited even with emerging developments. This study aimed to apply bionanomaterials (PRP/collagen scaffold) for human auricular neocartilage reconstruction by using microtia chondrocytes. The results showed that PRP (TGF-β1 750 pg/mL and 1 ng/mL) increased cell viability of microtia chondrocytes during in vitro 9-day cultures. Additionally, chondrogenic-specific mRNA of Aggrecan and type II collagen (Col II) was significantly and continuously expressed with PRP treatment during the 21-day in vitro expansion. Tissue engineering of auricular neocartilage was performed by seeding microtia chondrocytes in bionanomaterials (PRP/collagen scaffold) 3-dimensional (3D) cultures. Immunohistochemistry (IHC) of Col II showed intensive signals between cells and matrix after 4-week cultures. Conclusion. Our results demonstrated that PRP promotes proliferation and redifferentiation of microtia chondrocytes and provides regenerative potentials in auricular neocartilage reconstruction.

Aging is related to loss of functional stem cell accompanying loss of tissue and organ regeneration potentials. Previously, we demonstrated that the life span of ovariectomy-senescence accelerated mice (OVX-SAMP8) was significantly prolonged and similar to that of the congenic senescence-resistant strain of mice after platelet rich plasma (PRP)/embryonic fibroblast transplantation. The aim of this study is to investigate the potential of PRP for recovering cellular potential from senescence and then delaying animal aging. We first examined whether stem cells would be senescent in aged mice compared to young mice. Primary adipose derived stem cells (ADSCs) and bone marrow derived stem cells (BMSCs) were harvested from young and aged mice, and found that cell senescence was strongly correlated to animal aging. Subsequently, we demonstrated that PRP could recover cell potential from senescence, such as promote cell growth (cell proliferation and colony formation), increase osteogenesis, decrease adipogenesis, restore cell senescence related markers and resist the oxidative stress in stem cells from aged mice. The results also showed that PRP treatment in aged mice could delay mice aging as indicated by survival, body weight and aging phenotypes (behavior and gross morphology) in term of recovering the cellular potential of their stem cells compared to the results on aged control mice. In conclusion these findings showed that PRP has potential to delay aging through the recovery of stem cell senescence and could be used as an alternative medicine for tissue regeneration and future rejuvenation.

SUMMARY Clinically, Gore-Tex Expanded-Polytetrafluoroethylene™ (E-PTFE™) has been used to reconstruct the lateral temporomandibular joint (TMJ) ligament. The purpose of this study was to assess changes in the biomechanical properties of implanted E-PTFE™ over time with respect to tissue infiltration. Ninety-six specimens of implants were divided into four groups. Group A was the experimental group. Thirty-six autoclave-sterilized specimens were subcutaneously implanted into the backs of 36 rats. The rats were randomly sacrificed at 2 (n= 12), 7 (n= 12) and 12 (n = 12) weeks. The implants were tested for mechanical properties including maximal stress, strain and Young's modulus of elasticity (E) using the servo-hydraulic material testing system (MTS). Group B was the in vitro control group. Thirty-six specimens were placed in tissue culture media at 37 °C for a time period equivalent to the experimental group to simulate the effect of a moist, warm environment on biomechanical properties. Group C was the temperature and pressure control group. Twelve specimens were autoclave-sterilized to determine the changes of tensile strength under high temperature and pressure. Control group D (no treatment) was tested to determine the initial tensile strength. The results showed significantly larger maximal stress as well as an increase in E and smaller maximal strain in experimental group A than in control groups B, C and D. There was no significant difference among control groups B, C and D. Histological examination of implants at 12 weeks demonstrated that 0·2–0·3 mm of 1-mm thick implants were occupied by connective tissue from each side. It may be concluded that E-PTFE™ implants become stronger and less flexible after implantation in vivo.

Low-back pain caused by intervertebral disc degeneration could be recovered by the regeneration of the nucleus pulposus (NP). This study aimed to establish a chondrogenic recovery model with promising a human NP (hNP) cell line, an immortalized hNP (ihNP), which could be a screening platform to identify regenerative drugs. The ihNP cells were created from primary human NP cells transfected with a retroviral vector-driven HPV16 E6/E7. Growth properties and characteristics of ihNP were evaluated by comparing with parental NP cells. Successful immortalization of ihNP cells stably expressed HPV 16 E6/E7 mRNA. The doubling time of ihNP was shortened to 53.16±2.63 h compared with parental hNP-P1. Cell cycle regulators, including p53, p21, and pRB were downregulated compared to parental hNP-P1. The in vivo neoplastic forming assay also demonstrated that the ihNP was nontumorigenic. After 25 generations of cell cultures, the ihNP cells, yet stably expressed chondrogenic genes, including (SOX9), type II collagen (Col II), aggrecan, decorin, biglycan, and versican. Higher expressions of chondrogenic proteins, including Col II, phosphorylated SOX9 (p-SOX9), and CD44 were also determined. Under the stressful inflammatory conditions induced by lipopolysaccharides (LPS), the regenerative and anti-inflammatory potentials of ihNP in two-dimensional culture with the presence of platelet-rich plasma (PRP) were evaluated by reverse transcriptase polymerase chain reaction. PRP showed significant effects on restoring diminished chondrogenic markers and deleterious inflammatory responses induced by LPS in ihNP. The therapeutic potentials of ihNP in three-dimensional neocartilage model could also be exerted by PRP using histological evaluation and immunological staining. Hence, the established ihNP cells can provide a chondrogenic recovery model as a regenerative drug screening tool for further regenerative drug discovery and development.

Osteoarthritis (OA) is a common rheumatic disease associated with imbalanced cartilage homeostasis which could be corrected by mesenchymal stem cells (MSCs) therapy. However, MSCs from different origins might exhibit distinct differentiation capacities. This study was undertaken to compare the therapeutic efficacies between MSCs from cord blood (CB-MSCs) and bone marrow (BM-MSCs) on OA treatment. The surface phenotypes and multipotent capacities of CB-MSCs and BM-MSCs were first characterized. The coculture commitment system was subsequently utilized for comparing the patterned molecules in stage-specific chondrogenesis of committed MSCs. For examining the therapeutic efficacies, committed CB-MSCs and BM-MSCs were encapsulated in neo-cartilage and subjected into pro-inflammatory cytokine environment. Finally, chondrogenic and inflammatory cytokine profiles in committed MSCs were evaluated. CB-MSCs and BM-MSCs were both negative for hematopoietic markers and positive for adhesion and mesenchymal cell markers. The CB-MSCs showed a markedly higher chondrogenic potential and relatively lower osteogenic and adipogenic capacities than BM-MSCs. During chondrogenesis, the committed CB-MSCs also showed significant increases in cell proliferation, adhesion molecules, signaling molecules, and chondrogenic-specific gene expressions in a coculture system. For the therapeutic efficacies, the committed CB-MSCs could strongly recover the pro-inflammatory cytokines diminished-Col II and proteoglycan expressions in a 3D arthritic model. The IL-10, ICAM-1 and TGF-β1 were also up-regulated in committed CB-MSCs analyzed by using cytokine profiling. Our data demonstrate that CB-MSCs possess specific advantages in cartilage regeneration over BM-MSCs. The CB-MSCs showed a better therapeutic potential that can contribute to advanced cell-based transplantation for clinical OA therapy.

The aim of this study was to develop a new diagnostic and therapeutic approach for the treatment of osteoporosis. Previously, we demonstrated that intraosseous transplantation of platelet-rich plasma (PRP) treated-osteoblast-like cells into ovariectomized senescence-accelerated mice (OVX-SAMP8) prevented the development of osteoporosis. In continuation, we aimed to explore the complex etiology of osteoporosis using this platform. An inverse relationship between bone marrow adipogenesis and osteogenesis has been suggested in the development of osteoporosis but the underlying mechanisms remain poorly described. To address these issues, we used PRP to inhibit adipocyte differentiation by promoting osteoblastic differentiation in adipocytes. In addition, a positive correlation between an increase in bone marrow adipocytes and bone loss was established. We assessed this relationship using an osteoporotic animal disease model which consisted of young (for prevention) and old (for treatment) OVX-SAMP8 mice. This animal model demonstrated that PRP treatment mainly exerted its action via promoting bone regeneration but also appeared to suppress adipogenesis within the marrow. The findings and methodology of this study could potentially be applied in the prevention and treatment of osteoporosis.

Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of α-tubulin with β-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with α-tubulin and β-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of α-tubulin and β-tubulin, increased α-tubulin and β-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

We aim to establish a 3D model of cartilage wound healing, and explore the involvement of chondrocytes in its repair. To characterize chondrocyte involvement in wound healing, an in vitro 3D model composed of chondrocyte mixing with either type II/I collagen or type I collagen matrix was established. The "defects" measuring 5 mm in diameter were made on each collagen matrix-chondrocyte construct to mimic in vivo cartilage defects. The effects of basic fibroblast growth factor (bFGF) on chondrocytes migration and differentiation were studied. The migration and Glucosaminoglycan (GAG) synthesis of chondrocytes in the defect areas were observed by microscopy after Alcian-blue staining. In the presence of bFGF, GAG expression increased significantly when chondrocytes were cultured in type II/I collagen matrix compared to type I collagen matrix. However, mild GAG accumulation was also found when cells were cultured in either type I or type II/I collagens without bFGF. In a 3D model of cartilage wound healing, bFGF promote chondrocyte proliferation, migration and differentiation in the presence of type II/I collagen matrix, and showed potential to regulate wound healing. These wound healing models may provide feasible methods to explore various drugs prior to human trials.

Objective Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)–based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration. Methods A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability. Results Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor β superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold. Conclusion These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell-based transplantation for future clinical use.

This aim of our study was to evaluate a novel cell-based therapy for contusion spinal cord injury (SCI) using embryonic-derived NIH3T3 cells, which endogenously express glial cell line-derived neurotrophic factor (GDNF).Proliferation and differentiation of transplanted NIH3T3 cells and their anti-apoptotic effects were examined after their engraftment into the spinal cords of Long-Evans rats subjected to acute SCI at the T10 vertebral level by a New York University impactor device. NIH3T3 cells were initially engineered to contain dual reporter genes, namely thymidine kinase (T) and enhanced green fluorescence protein (G), for in vivo cell tracking by both nuclear and fluorescence imaging modalities.Planar and fluorescence imaging demonstrated that transplanted NIH3T3-TG cells at the L1 vertebral level migrated 2 cm distal to the injury site as early as 2 h, and the signals persisted for 48 h after SCI. The expression of GDNF by NIH3T3-TG cells was then confirmed by immunohistochemical analysis both in vitro and in vivo. GDNF-secreting NIH3T3-TG transplant provided anti-apoptotic effects in the injured cord over the period of 3 wk. Finally, NIH3T3-TG cells cultured under neuronal differentiation medium exhibited both morphologic and genetic resemblance to neuronal cells.GDNF-secreting NIH3T3-TG cells in combination with molecular imaging could be a platform for developing therapeutic tools for acute SCI.

Chondrocytes are useful as a cell culture system for studying arthritic degeneration in tissue engineered cartilage. However, primary chondrocytes have short in vitro lifespan and rapid shift of collagen phenotype. In this study, we used a high dosage of retroviral vector LXSN16E6E7 to transduce human primary chondrocytes and obtained an actively proliferating cell line, designated hPi, which expresses HPV-16 E6/E7 mRNA in early passages. Parental primary chondrocytes cease to grow after five passages, whereas hPi could be propagated beyond 100 passages without requiring additional cell elements in defined medium. After 48 passages, hPi can also give many profiles similar to those of parental primary chondrocyte, including type II collagen in mRNA and protein level, aggrecan in mRNA level, lacunae in type I collagen matrices, and morphology with GAG-specific Alcian blue staining. hPi has shown neoplastic transformation, as examined by NOD-SCID mice tumorgenicity assays for 3 months. Our results indicated that human primary chondrocytes could be immortalized by transduction with HPV-16 E6/E7, preserving stable cartilage-specific differentiation markers. The established chondrocyte cell line could provide a novel model to engineer cartilage in vitro and in vivo for cartilage repair research and clinical application. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.

Background Current treatments for osteoporosis are associated with various side effects and do not prevent the age-related decrease in osteoblast number. The objective of this study was to evaluate the effects of iQPR-H2O on osteogenesis. Methods Mouse fibroblast NIH3T3 and pre-osteoblastic MC3T3-E1 cells were cultured in medium prepared with iQPR-H2O or unprocessed mineral water (control cells), and proliferation and differentiation were assessed by MTT and alkaline phosphatase assay, respectively. Mineral deposition by the cells was determined using Alizarin red S staining. A mouse model of osteoporosis, ovariectomized SAMP8 mice, was used to evaluate the effects of iQPR-H2O on osteogenesis in vivo. Mice were given either iQPR-H2O or unprocessed mineral water (control group) for four months after which bone mass density (BMD) measurements were made using a bone densitometer and hematoxylin and eosin staining of bone samples. Results NIH3T3 cells grown in medium prepared with iQPR-H2O exhibited significantly greater proliferation. NIH3T3 and MC3T3-E1 cells demonstrated a significant increase in alkaline phosphatase levels in the iQPR-H2O group. MC3T3-E1 cells showed mineralization at day 28. mRNA expression levels of both osteopontin and runt-related transcription factor 2 in MC3T3-E1 cells were higher in the iQPR-H2O group compared with the control group. After four months, significantly greater bone regeneration was evident in ovariectomized SAMP8 mice administered iQPR-H2O as compared with control group. Conclusions iQPR-H2O may reduce the symptoms of osteoporosis by improving osteogenesis.