Characterization of temporomandibular joint articular disc progenitor cell clones.

A critical component of the temporomandibular joint (TMJ) is the fibrocartilage articular disc (AD). Researchers have attempted to regenerate the AD to alleviate TMJ osteoarthritis but alternative cell sources for use in AD regenerative approaches are needed due to insufficient extracellular matrix (ECM) production by total articular disc cells (TACs). Tissue-specific progenitor cells have been identified in many tissues. The aim of the present study was to identify adult multipotent progenitor cells within the AD suitable for regenerative medicine applications. A novel AD progenitor cell population was identified in rhesus macaques. Clonally derived articular disc progenitor cells (ADPs) were isolated using fibronectin differential cell adhesion. ADPs represent between 1 and 3 % of the TAC population and are capable of in vitro expansion beyond 60 population doublings. ADPs were characterized using osteogenic, adipogenic, and fibrochondrogenesis differentiation assays. Clones exhibited phenotypic plasticity, differentiating into osteocytes, adipocytes, and fibrochondrocytes. ECM secretion profiles following fibrochondrogenic differentiation were assessed using immunohistochemistry (IHC), fluorescently activated cell sorting (FACS), total collagen, and glycosaminoglycan (GAG) assays and compared with TACs, articular cartilage progenitor cells (ACPs), tendon progenitor cells (TPCs) and bone-marrow-derived mesenchymal stem cells (BMMSCs). ADP pellet cultures produced a biochemical phenotype similar to native AD tissue, with production of versican (VCAN) and collagen types I, II, III, and VI (COL1, COL2, COL3, COL6). However, clonally derived ADP cell lines produced different amounts of ECM and exhibited different expansion potentials. These findings indicated flexibility in clone selection for potential regenerative strategies to recapitulate native anisotropy.

Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Cells under pressure - the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis.

Early osteoarthritis (OA), characterised by cartilage defects, is a degenerative disease that greatly affects the adult population. Cell-based tissue engineering methods are being explored as a solution for the treatment of these chondral defects. Chondrocytes are already in clinical use but other cell types with chondrogenic properties, such as mesenchymal stem cells (MSCs), are being researched. However, present methods for differentiating these cells into stable articular-cartilage chondrocytes that contribute to joint regeneration are not effective, despite extensive investigation. Environmental stimuli, such as mechanical forces, influence chondrogenic response and are beneficial with respect to matrix formation. In vivo, the cartilage is subjected to multiaxial loading involving compressive, tensile, shear and fluid flow and cellular response. Tissue formation mechanobiology is being intensively studied in the cartilage tissue-engineering research field. The study of the effects of hydrostatic pressure on cartilage formation belongs to the large area of mechanobiology. During cartilage loading, interstitial fluid is pressurised and the surrounding matrix delays pressure loss by reducing fluid flow rate from pressurised regions. This fluid pressurisation is known as hydrostatic pressure, where a uniform stress around the cell occurs without cellular deformation. In vitro studies, examining chondrocytes under hydrostatic pressure, have described its anabolic effect and similar studies have evaluated the effect of hydrostatic pressure on MSC chondrogenesis. The present review summarises the results of these studies and discusses the mechanisms through which hydrostatic pressure exerts its effects.

Mechanosensitive MiRs regulated by anabolic and catabolic loading of human cartilage.

OBJECTIVE: Elucidation of whether miRs are involved in mechanotransduction pathways by which cartilage is maintained or disturbed has a particular importance in our understanding of osteoarthritis (OA) pathophysiology. The aim was to investigate whether mechanical loading influences global miR-expression in human chondrocytes and to identify mechanosensitive miRs responding to beneficial and non-beneficial loading regimes as potential to obtain valuable diagnostic or therapeutic targets to advance OA-treatment. METHOD: Mature tissue-engineered human cartilage was subjected to two distinct loading regimes either stimulating or suppressing proteoglycan-synthesis, before global miR microarray analysis. Promising candidate miRs were selected, re-evaluated by qRT-PCR and tested for expression in human healthy vs OA cartilage samples. RESULTS: After anabolic loading, miR microarray profiling revealed minor changes in miR-expression while catabolic stimulation produced a significant regulation of 80 miRs with a clear separation of control and compressed samples by hierarchical clustering. Cross-testing of selected miRs revealed that miR-221, miR-6872-3p, miR-6723-5p were upregulated by both loading conditions while others (miR-199b-5p, miR-1229-5p, miR-1275, miR-4459, miR-6891-5p, miR-7150) responded specifically after catabolic loading. Mechanosensitivity of miR-221 correlated with pERK1/2-activation induced by both loading conditions. The miR-response to loading was transient and a constitutive deregulation of mechano-miRs in OA vs healthy articular cartilage was not observed. CONCLUSIONS: MiRs with broader vs narrower mechanosensitivity were discovered and the first group of mechanosensitive miRs characteristic for non-beneficial loading was defined that may shape the proteome differentially when cartilage tissue is disturbed. The findings prompt future investigations into miR-relevance for mechano-responsive pathways and the corresponding miR-target molecules.

Reoxygenation enhances tumour necrosis factor alpha-induced degradation of the extracellular matrix produced by chondrogenic cells.

Mesenchymal stem cells (MSCs) have been considered as a potential source for cell-based therapies in arthritic diseases for both their chondrogenic and anti-inflammatory properties. Thus, we examined how MSC-based neocartilage responds to tumour necrosis factor alpha (TNF-α) compared to articular chondrocyte (AC)-based neocartilage. Since oxygen tension is altered in arthritic joints, we also examined how increased oxygen tension influences this process. Monolayer-expanded healthy human ACs and bone marrow MSCs were cultured in chondrogenic medium in three-dimensional culture under hypoxia. They were then exposed to TNF-α under hypoxic or increased oxygen tension. We found no inherent anti-inflammatory potential of MSC-derived neocartilage as it pertains to the enzymes studied here: more degradative enzymes were upregulated by TNF-α in MSCs than in ACs, regardless of the oxygen tension. MSCs were also more sensitive to reoxygenation during TNF-α exposure, as indicated by increased proteoglycan loss, increased aggrecanase-generated metabolites, and further upregulation of the major aggrecanases, ADAMTS4 and ADAMTS5. There was also evidence of matrix metalloproteinase (MMP)-mediated aggrecan interglobular domain cleavage and type II collagen loss in response to TNF-α in both MSCs and ACs, but more MMPs were further upregulated by reoxygenation in MSCs than in ACs. Our study provides further evidence that consideration of oxygen tension is essential for studying cartilage degradation; for example, neocartilage produced from MSCs may be more sensitive to the negative effects of repeated hypoxia/reoxygenation events than AC-derived neocartilage. Consideration of the differences in responses may be important for cell-based therapies and selection of adjunctive chondroprotective agents.

Caffeic acid phenethyl ester extends survival of a mouse model of amyotrophic lateral sclerosis.

There is currently very limited effective pharmacological treatment for amyotrophic lateral sclerosis. Recent evidence suggests that caffeic acid phenethyl ester has strong anti-inflammatory, anti-oxidative, and anti-neuronal death properties; thus, the present study tested the effects of caffeic acid phenethyl ester in mice expressing a mutant superoxide dismutase (SOD1(G93A)) linked to human amyotrophic lateral sclerosis. Administration of caffeic acid phenethyl ester after symptom onset significantly increased the post-onset survival and lifespan of SOD1(G93A) mice. Moreover, immunohistochemical analysis detected less activation of microglia and astrocytes and higher motor neuron counts at an early symptomatic stage (7 days following onset) in the spinal cords of SOD1(G93A) mice given caffeic acid phenethyl ester treatment. Additionally, lower levels of phosphorylated p38, a mitogen-activated protein kinase that is involved in both inflammation and neuronal death, were observed in the spinal cords of SOD1(G93A) mice treated with caffeic acid phenethyl ester for 7 days. These results indicate that caffeic acid phenethyl ester may represent a novel and effective therapeutic for the treatment of amyotrophic lateral sclerosis, and these significant neuroprotective effects observed in a commonly used amyotrophic lateral sclerosis mouse model validate the therapeutic potential of caffeic acid phenethyl ester for slowing disease progression by attenuating the neuroinflammation and motor neuron cell death associated with clinical amyotrophic lateral sclerosis pathology.

Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels.

Biological activity can be added to synthetic scaffolds by incorporating functional peptide sequences that provide enzyme-mediated degradation sites, facilitate cellular adhesion or stimulate signaling pathways. Poly(ethylene glycol) diacrylate is a popular synthetic base for tissue engineering scaffolds because it creates a hydrophilic environment that can be chemically manipulated to add this biological functionality. Furthermore, the acrylate groups allow for encapsulation of cells using photopolymerization under physiological conditions. One complication with the addition of these peptides is that aromatic amino acids absorb light at 285 nm and compete with the ultraviolet (UV)-sensitive photoinitiators such as IrgacureTM 2959 (I2959), the most commonly used initiator for cytocompatible photoencapsulation of cells into synthetic scaffolds. In this study we define non-toxic conditions for photoencapsulation of human mesenchymal stem cells (hMSC) in PEGDA scaffolds using a visible light photoinitiator system composed of eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone. This visible light photoinitiator produced hydrogel scaffolds with an increased viability of encapsulated hMSCs and a more tightly crosslinked network in one-third the time of UV polymerization with I2959.

Tuberculous dactylitis--an easily missed diagnosis.

The prevalence of tuberculosis (TB) continues to rise worldwide. Current migration patterns and increased travel to high-prevalence TB countries will result in more frequent presentations of less common forms of TB. Tuberculous dactylitis, a form of tuberculous osteomyelitis, is well recognised in countries with a high prevalence of TB. We provide a systematic review of all published cases of tuberculous dactylitis in children and adolescents and describe a case to illustrate the typical features of the disease. Our review revealed 37 cases of tuberculous dactylitis in children and adolescents, all reported in the last 17 years. Children less than 10 years of age are most frequently affected and the hand is the most commonly affected site. Concurrent pulmonary TB is present in a fifth of cases and systemic symptoms are usually absent. Positive TST and IGRA support the presumptive diagnosis, but cannot be used as rule-out tests. The definitive diagnosis relies on the detection M. tuberculosis by PCR or culture. Treatment should comprise of a standard three to four drug anti-tuberculous regimen. The optimal treatment duration remains unknown. Surgery has a limited role in the treatment in general but may play a supportive role, and curettage of the cavity has been recommended for avascular lesions.

AMP-activated kinase mediates adipose stem cell-stimulated neuritogenesis of PC12 cells.

Adipose tissue stroma contains a population of mesenchymal stem cells, which support repair of damaged tissues through the protective effects of secreted trophic factors. Neurotrophic factors, including nerve growth factor (NGF) have been identified in media collected from cultured adipose-derived stem cells (ASC). We previously demonstrated that administration of cell-free ASC conditioned medium (ASC-CM) at 24 h after injury reduced lesion volume and promoted functional recovery in a rat model of neonatal brain hypoxic-ischemic (HI) injury. The timing of administration well after the peak in neural cell apoptosis in the affected region suggests that regeneration of lost neurons is promoted by factors in ASC-CM. In this study, we determined which of the factors in ASC-CM could induce neurogenesis by testing the ability of the mixture, either whole or after inactivating specific components, to stimulate neurite outgrowth in vitro using the neurogenic cell line PC12. Neuritogenesis in PC12 cells treated with ASC-CM was observed at a level comparable to that observed with purified recombinant NGF. It was observed that NGF in ASC-CM was mainly responsible for inducing PC12 cell neuritogenesis. Interestingly, both ASC-CM and NGF induced PC12 cell neuritogenesis through activation of the AMP-activated kinase (AMPK) pathway which is the central protein involved in controlling many critical functions in response to changes in the cellular energy status. Pharmacological and genetic inhibition of AMPK activity greatly reduced neuritogenesis in PC12 cells. These results suggest that, in addition to possessing neuroprotective properties, ASC-CM mediates repair of damaged tissues through inducing neuronal differentiation via NGF-induced AMPK activation.

Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis.

Transplantation of pluripotent adipose stem/stromal cells (ASC) alleviates tissue damage and improves functional deficits in both stroke and cardiovascular disease animal models. Recent studies indicate that the primary mechanism of ASC-induced repair may not be directly related to tissue regeneration through differentiation, but rather through paracrine mechanisms provided by secreted pro-survival and repair-inducing trophic factors. In this study, we have found that ASC-conditioned medium (ASC-CM) potently protected cerebellar granule neurons (CGN) from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by IGF-1. These data suggest that of the many neuroprotective factors secreted by ASC, IGF-1 is the major factor that mediates protection against serum and potassium deprivation-induced CGN apoptosis. This study establishes a mechanistic basis supporting the therapeutic application of ASC for neurological disorders, specifically through paracrine support provided by trophic factor secretion.

Adipose stromal cells-conditional medium protected glutamate-induced CGNs neuronal death by BDNF.

The delivery of factors secreted by adipose stromal cells (ASC) to the brain may be a viable neuroprotective therapeutic option. In this study, we investigated the bioactivity of ASC-conditioned medium (ASC-CM) in glutamate-induced neurotoxicity and found that the ASC-CM significantly blocked glutamate neurotoxicity. We identified the brain-derived neurotrophic factor (BDNF) in the ASC-CM by using Western blot and demonstrated that this activity was critical for the neuroprotective effect of ASC-CM in excytotoxicity models. Furthermore, inactivating BDNF also attenuated the suppression by ASC-CM of glutamate-induced caspase-3 activity, but not p38 phosphorylation. These findings suggest that among ASC secrete a potent combination of factors, BDNF play a major role in neuroprotection against excytotoxicity.

Caffeic acid phenethyl ester prevents cerebellar granule neurons (CGNs) against glutamate-induced neurotoxicity.

Caffeic acid phenethyl ester (CAPE) is an active component of propolis obtained from honeybee hives and is found to have the following properties: anti-mitogenic, anti-carcinogenic, anti-inflammatory, immunomodulatory, and antioxidant. Recent reports suggest that CAPE also has a neuronal protective property against ischemic injury. Since excitotoxicity may play an important role in ischemia, in this study, we investigated whether CAPE could directly protect neurons against excitotoxic insult. We treated cultured rat cerebellar granule neurons (CGNs) with excitotoxic concentrations of glutamate in the presence or absence of CAPE and found that CAPE markedly protected neurons against glutamate-induced neuronal death in a concentration-dependent fashion. Glutamate-induced CGNs death is associated with time-dependent activation of caspase-3 and phosphorylation of p38, both events of which can be blocked by CAPE. Treating CGNs with specific inhibitors of these two enzymes together exerts a synergistic neuroprotective effect, similar to the neuroprotective effect of CAPE exposure. These results suggest that CAPE is able to block glutamate-induced excitotoxicity by inhibiting phosphorylation of p38 and caspase-3 activation. This finding may further help understanding of the mechanism of glutamate-induced neuronal death and CAPE-induced neuroprotection against excitotoxicity.

Randomized trial of botulinum toxin injections into the salivary glands to reduce drooling in children with neurological disorders.

The primary aim of this randomized, controlled trial was to assess the effectiveness of botulinum toxin A (BoNT-A) injections into the submandibular and parotid glands on drooling in children with cerebral palsy (CP) and other neurological disorders. Secondary aims were to ascertain the duration of any such effect and the timing of maximal response. Of the 48 participants (27 males, 21 females; mean age 11y 4mo [SD 3y 3mo], range 6-18y), 31 had a diagnosis of CP and 15 had a primary intellectual disability; 27 children were non-ambulant. Twenty-four children randomized to the treatment group received 25 units of BoNT-A into each parotid and submandibular gland. Those randomized to the control group received no treatment. The degree and impact of drooling was assessed by carers using the Drooling Impact Scale questionnaire at baseline and at monthly intervals up to 6 months postinjection/baseline, and again at 1 year. Maximal response was at 1 month at which time there was a highly significant difference in the mean scores between the groups. This difference remained statistically significant at 6 months. Four children failed to respond to the injections, four had mediocre results, and 16 had good results. While the use of BoNT-A can help to manage drooling in many children with neurological disorders, further research is needed to fully understand the range of responses.

Ascorbate-enhanced chondrogenesis of ATDC5 cells.

The ATDC5 cell line exhibits the multistep chondrogenic differentiation observed during endochondral bone formation. However, it takes up to two months to complete the process of cell expansion, insulin addition to promote differentiation and further changes in culture conditions effectively to induce hypertrophy. We sought to produce consistent chondrogenesis with significant hypertrophic differentiation with simpler conditions in a more practical time period. By adding ascorbate, the prechondrogenic proliferation phase was shortened from 21 to 7 days, with production of cartilaginous nodules during the chondrogenic phase, after insulin addition, that were greater in number and larger in size. Immunohistochemistry indicated much greater matrix elaboration and the mRNA expression of sox9, aggrecan and collagen type II were all significantly increased earlier and to a much higher degree when compared with controls. Moreover, there was a robust induction of hypertrophy: Col10a1, Runx2 and Mmp13 were all induced within 7-10 days. In conclusion, addition of ascorbate to ATDC5 cultures shortened the prechondrogenic proliferation phase, produced earlier chondrogenic differentiation, heightened gene expression and robust hypertrophic differentiation, abrogating the need for extended culture times and the changes in culture conditions. This simple modification considerably enhances the practicality of this cell line for studies of chondrogenesis.

The "U-ligament" for collateral ligament reconstruction in duplicate thumb.

Duplicate thumb is a common congenital difference with complex management. Surgical treatment includes reconstruction of the collateral ligaments to optimise future function and cosmesis. We present the use of the "U-ligament" to further refine collateral ligament reconstruction. We have found it to be a simple and reliable technique.

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro.

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow-derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1-7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non-loaded controls. In contrast, for the multi-day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.

Mesenchymal cell transfer for articular cartilage repair.

Mature articular cartilage has a poor reparative response to injury and its irreparable breakdown is the common feature of degenerative joint diseases. If articular cartilage lesions become symptomatic, the orthopaedic surgeon must decide on a treatment option. The treatment options include conversion of chondral lesions to osteochondral lesions, which facilitates migration of cells from the marrow space to effect repair. In recent years, a greater emphasis has been placed on tissue engineering strategies and thus several new treatment options have been introduced, including the use of cell transplantation. Several tissue sources and cell types can potentially be used for this type of therapy. These include autologous or allograft chondrocytes and mesenchymal progenitor cells from various tissues. These cells may be delivered to articular cartilage lesions by a variety of methods including direct cell injection to the lesion or seeding in a biodegradable scaffold prior to implantation. In this review, the potential of cell transplantation for articular cartilage repair and regeneration will be discussed. The authors will focus on the available technologies and the present limitations of cell-based therapies.

Proximal interphalangeal joint surface replacement arthroplasty.

A consecutive series of 20 joints in 13 patients underwent arthroplasty with the RMS PIP joint surface replacement implant. Twelve joints were treated for painful osteoarthritis (all females). Two joints were implanted for rheumatoid arthritis, two for post-traumatic pain and stiffness, two for post-traumatic stiffness and one each for post-traumatic pain and pain-free post-traumatic instability. Excellent, often total long-term pain relief was obtained in 18 joints. The other two patients with (compensible work-related) post-traumatic pain and stiffness reported "50-70% pain reduction". No patients lost movement and 14 out of 20 joints were pain-free with a 73.1 degrees average arc of motion. Six joints from the first half of the series had poor motion (average arc of 19.6 degrees ), even after open extensor tenolysis or manipulation under anaesthesia. As experience was gained, reliably better results were achieved with a more intensive regimen of hand therapy, particularly within the first post-operative week.

Ascending necrotising fasciitis as a result of odontogenic infection: a report of two cases.

Necrotising fasciitis is a severe soft tissue infection which spreads rapidly through fascial planes, is characterised by soft tissue necrosis and is potentially life-threatening. It is a rare entity in the head and neck region. The management of this condition is difficult and early diagnosis and aggressive surgical and medical management are essential. This paper reports two cases of necrotising fasciitis as a result of ascending odontogenic infection involving the temporalis muscle.

Primary shortening of the pollicized long flexor tendon in congenital pollicization.

Primary shortening of the flexor digitorum profundus tendon to the pollicized index finger was routinely performed during all congenital pollicizations over a 3-year period. Interphalangeal joint flexion of the pollicized digit was observed to begin within weeks after pollicization. The rebalancing of the pollicized long flexor musculotendinous unit has the potential for greater long-term strength.