Aiqun Wei

Apoptosis in rotator cuff tendonopathy.

The aim of this study was to investigate the involvement of apoptosis (programmed cell death) in the pathogenesis of rotator cuff disorders. The edges of torn supraspinatus rotator cuff tendons were collected from patients with rotator cuff tear (n = 25). Samples of the intra‐articular portion of subscapularis tendons were collected from patients without rotator cuff tear as control (n = 6). To minimize individual variance, we also collected six pairs of supraspinatus tendon and subscapularis tendon from six patients with rotator cuff tears. Apoptosis was detected by in situ DNA end labelling assay and DNA laddering assay. Immunohistochemical staining was performed to identify cells undergoing apoptosis. Control subscapularis tendon had normal morphology. Tendon from torn supraspinatus rotator cuff showed significant mucoid degeneration. Within the areas of degeneration, there were large numbers of apoptotic cells. The percentage of apoptotic cells in the degenerative rotator cuff (34%) was significantly higher than that in controls (13%) (p < 0.001). The excessive apoptosis detected in degenerative rotator cuff tissue was confirmed by DNA laddering assays. This is the first report of excessive apoptosis in degenerating rotator cuff tendon. Cells undergoing apoptosis in rotator cuff were mainly fibroblast‐like cells. These finding indicate that apoptosis may play an important role in the pathogenesis of rotator cuff degeneration.

Cytokines and apoptosis in supraspinatus tendinopathy

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010.

BMP-2 Enhances TGF-β3–Mediated Chondrogenic Differentiation of Human Bone Marrow Multipotent Mesenchymal Stromal Cells in Alginate Bead Culture

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.

The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discs

Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone‐marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc‐like cells. Human bone‐marrow derived CD34+ (hematopoietic progenitor cells) and CD34− (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent‐labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34− cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox‐9). In contrast, the fluorescent labeled CD34+ cells could not be detected after day 21. No fluorescence‐positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34− cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration.

Expression and regulation of peroxiredoxin 5 in human osteoarthritis.

Reactive oxygen species (ROS) are implicated in the pathogenesis of osteoarthritis (OA). However, little is known about the antioxidant defence system in articular cartilage. We investigated the expression and regulation of peroxiredoxin 5 (PRDX5), a newly discovered thioredoxin peroxidase, in human normal and osteoarthritic cartilage. Our results show that human cartilage constitutively expresses PRDX5. Moreover, the expression is up‐regulated in OA. Inflammatory cytokines tumour necrosis factor α and interleukin 1 β contribute to this up‐regulation by increasing intracellular ROS production. The present study suggests that PRDX5 may play a protective role against oxidative stress in human cartilage.

Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon.

We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi‐quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady‐state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23‐fold increase) at day 4, the endothelial isoform (eNOS) (24‐fold increase) at day 7 and the neuronal isoform (bNOS) (seven‐fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five‐fold increase in the NOS activity, as detected by 3H‐arginine to 3H‐citrulline conversion, at day 7 postinjury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.

The cell specific temporal expression of nitric oxide synthase isoforms during achilles tendon healing.

Abstract.Objective and design: We have previously shown that nitric oxide synthase (NOS) activity is upregulated following tendon injury, and that this activity is important to Achilles tendon healing. The aim of this study was to identify the cellular distribution of nitric oxide synthase isoforms during tendon healing.¶Material or subjects: Surgical division of the right Achilles tendon was performed in eighty-five male Sprague-Dawley rats. Healing Achilles tendons were harvested at 4, 7, 14 and 21 days following the surgery. The un-injured left Achilles tendons were used as controls. Using RNase protection assays, in situ hybridization and immunohistochemistry, mRNA and protein of NOS isoforms were evaluated.¶Results: Minimal NOS expression was found in un-injured tendon. A cell specific temporal pattern for the mRNA and protein for all three NOS isoforms was found following injury to the Achilles tendon. iNOS was maximal on day 4 in macrophages and fibroblasts. eNOS was maximal on day 4 in endothelial cells and fibroblasts. bNOS expression gradually increased up to day 21 and was found only in fibroblasts.¶Conclusions: These results suggest that all three nitric oxide synthase isoforms are expressed by fibroblasts in a coordinated temporal sequence during tendon healing. The sequential pattern of NOS expression in healing fibroblasts suggests that each NOS isoform may play a different role in the healing process and provides opportunities to modify tendon healing in the clinical setting.

Enhanced delivery of synthetic oligonucleotides to human leukaemic cells by liposomes and immunoliposomes

The ability of pH-sensitive liposomes and immunoliposomes to deliver synthetic antisense oligonucleotides (oligos) into human myeloid and lymphoid leukaemia cells was examined. The cellular uptake of an 18mer anti-myb oligonucleotide encapsulated in liposomes was from three- to five-fold higher than that of 32P-oligos alone. In addition, anti-CD32 or anti-CD2 immunoliposomes improved the delivery of oligos to leukaemic cells carrying the appropriate receptor for the specific antibody-linked immunoliposome. The uptake of oligos was twice that of the liposome or non-specific immunoliposome encapsulated oligos. These findings support the use of liposomes or immunoliposomes to deliver antisense oligos into human leukaemic cells.

Posterolateral intertransverse spinal fusion possible in osteoporotic rats with BMP-7 in a higher dose delivered on a composite carrier.

Study Design. Posterolateral intertransverse process spinal fusion (PLF) was performed in ovariectomized female rats using recombinant human BMP-7 (OP-1) delivered on a composite carrier. Objective. To investigate whether BMP-7 collagen on a composite carrier in a higher dose will enhance posterolateral spinal fusion in an estrogen deficiency rat model. Summary of Background Data. Osteoporosis is a systemic disease characterized by bone remodeling skewed in favor of excess bone resorption. This makes new bone formation and fixation of metallic implants difficult. Thus, treating osteoporotic patients who require posterior spinal fusion is challenging. Ovariectomized rats have been used as an osteoporotic model for posterolateral intertransverse process fusion. We have demonstrated in the past that endochondral bone formation in osteoporotic rats is delayed when compared with rats without osteoporosis. We have also shown that OP-1 Putty (BMP-7, collagen, and carboxy-methyl-cellulose) can overcome the effects of osteoporosis in a rat fracture model. However, it has not yet been demonstrated whether BMP-7 collagen composite carrier (Calstrux) can achieve a fusion in a process spinal fusion model in osteoporotic bone. Methods. A total of 42 ovariectomized Sprague-Dawley female rats were randomly assigned to 4 control and 2 experimental groups: (1) no Calstrux, no BMP; (2) 400 mg Calstrux alone; (3) 30 &mgr;g lactose + 400 mg Calstrux; (4) 90 &mgr;g lactose + 400 mg Calstrux; (5) 30 &mgr;g rhBMP-7 + 400 mg Calstrux; and (6) 90 &mgr;g rhBMP-7 + 400 mg Calstrux. Spinal fusion was evaluated by manual motion testing, microradiographs, computed tomographic scans, DEXA scans, and histology. Results. Ovariectomized rats receiving Calstrux alone or either dose of lactose and Calstrux did not show spinal fusion. Ovariectomized rats receiving 90 &mgr;g BMP-7 + 400 mg Calstrux showed significantly higher fusion rates than these control animals. (P < 0.0001). The rats receiving 30 &mgr;g BMP-7 + 400 mg Calstrux exhibited only partial fusion. Conclusion. BMP-7, delivered on a composite carrier, is able to overcome the detrimental effects of estrogen deficiency on posterolateral spinal fusion and generate a relatively robust fusion. The effect seems to be dose dependent.