Towards understanding how bisphosphonate-dependent alterations to nutrient canal integrity can contribute to risk for atypical femoral fractures: Biomechanical considerations and potential relationship to a real-world analogy.

Bisphosphonates are a class of drugs which have shown good efficacy in the treatment of post-menopausal osteoporosis, as well as a good safety profile. However, side-effects such as risk for atypical femoral fractures (AFF) have appeared, leading to a decline in use of the drugs by many patients who would benefit from the treatment. While patient characteristics have contributed to improved understanding of risk factors, the mechanisms involved that explain AFF risk have not appeared. Recently, the possibility that the mechanism(s) involved drug-induced modification of cells of the nutrient canals of the femur and subsequent compromise in the bone matrix has been published. The present Hypothesis article builds on the concept presented earlier and expands into biomechanical considerations. An analogy of the mechanisms involved to a real-life scenario is also presented. While this analogy has limitations, consideration of the biomechanical implications of progressive alterations to defects presented by compromised nutrient canal-bone matrix also presents potential relationships with AFF risk.

Are secondary effects of bisphosphonates on the vascular system of bone contributing to increased risk for atypical femoral fractures in osteoporosis?

Osteoporosis (OP) is a bone disease which affects a number of post-menopausal females and puts many at risk for fractures. A large number of patients are taking bisphosphonates (BPs) to treat their OP and a rare complication is the development of atypical femoral fractures (AFF). No real explanations for the mechanisms underlying the basis for development of where AFF develop while on BPs has emerged. The present hypothesis will discuss the possibility that part of the risk for an AFF is a secondary effect of BPs on a subset of vascular cells in a genetically at-risk population, leading to localized deregulation of the endothelial cell (EC)-bone cell-matrix units in nutrient channels/canals of the femur and increased risk for AFF. This concept of targeting ECs is consistent with location of AFF in the femur, the bilateral risk for occurrence of AFF, and the requirement for long term exposure to the drugs.

Effect of prebiotic fiber on physical function and gut microbiota in adults, mostly women, with knee osteoarthritis and obesity: a randomized controlled trial.

PURPOSE: Obesity is a primary risk factor for knee osteoarthritis (OA). Prebiotics enhance beneficial gut microbes and can reduce body fat and inflammation. Our objective was to examine if a 6-month prebiotic intervention improved physical function in adults with knee osteoarthritis and obesity. We also measured knee pain, body composition, quality of life, gut microbiota, inflammatory markers, and serum metabolomics. METHODS: Adults (n = 54, mostly women) with co-morbid obesity (BMI > 30 kg/m2) and unilateral/bilateral knee OA were randomly assigned to prebiotic (oligofructose-enriched inulin; 16 g/day; n = 31) or isocaloric placebo (maltodextrin; n = 21) for 6 months. Performance based-tests, knee pain, quality of life, serum metabolomics and inflammatory markers, and fecal microbiota and short-chain fatty acids were assessed. RESULTS: Significant between group differences were detected for the change in timed-up-and-go test, 40 m fast paced walk test, and hand grip strength test from baseline that favored prebiotic over placebo. Prebiotic also reduced trunk fat mass (kg) at 6 months and trunk fat (%) at 3 months compared to placebo. There was a trend (p = 0.059) for reduced knee pain at 6 months with prebiotic versus placebo. In gut microbiota analysis, a total of 37 amplicon sequence variants differed between groups. Bifidobacterium abundance was positively correlated with distance walked in the 6-min walk test and hand grip strength. At 6 months, there was a significant separation of serum metabolites between groups with upregulation of phenylalanine and tyrosine metabolism with prebiotic. CONCLUSION: Prebiotics may hold promise for conservative management of knee osteoarthritis in adults with obesity and larger trials are warranted. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov/study/NCT04172688.

eEF2 improves dense connective tissue repair and healing outcome by regulating cellular death, autophagy, apoptosis, proliferation and migration.

Outcomes following human dense connective tissue (DCT) repair are often variable and suboptimal, resulting in compromised function and development of chronic painful degenerative diseases. Moreover, biomarkers and mechanisms that guide good clinical outcomes after DCT injuries are mostly unknown. Here, we characterize the proteomic landscape of DCT repair following human Achilles tendon rupture and its association with long-term patient-reported outcomes. Moreover, the potential regulatory mechanisms of relevant biomarkers were assessed partly by gene silencing experiments. A mass-spectrometry based proteomic approach quantified a large number (769) of proteins, including 51 differentially expressed proteins among 20 good versus 20 poor outcome patients. A novel biomarker, elongation factor-2 (eEF2) was identified as being strongly prognostic of the 1-year clinical outcome. Further bioinformatic and experimental investigation revealed that eEF2 positively regulated autophagy, cell proliferation and migration, as well as reduced cell death and apoptosis, leading to improved DCT repair and outcomes. Findings of eEF2 as novel prognostic biomarker could pave the way for new targeted treatments to improve healing outcomes after DCT injuries.Trial registration: NCT02318472 registered 17 December 2014 and NCT01317160 registered 17 March 2011, with URL http://clinicaltrials.gov/ct2/show/NCT02318472 and http://clinicaltrials.gov/ct2/show/study/NCT01317160 .

The Heterogeneity of Post-Menopausal Disease Risk: Could the Basis for Why Only Subsets of Females Are Affected Be Due to a Reversible Epigenetic Modification System Associated with Puberty, Menstrual Cycles, Pregnancy and Lactation, and, Ultimately, Menopause?

For much of human evolution, the average lifespan was <40 years, due in part to disease, infant mortality, predators, food insecurity, and, for females, complications of childbirth. Thus, for much of evolution, many females did not reach the age of menopause (45-50 years of age) and it is mainly in the past several hundred years that the lifespan has been extended to >75 years, primarily due to public health advances, medical interventions, antibiotics, and nutrition. Therefore, the underlying biological mechanisms responsible for disease risk following menopause must have evolved during the complex processes leading to Homo sapiens to serve functions in the pre-menopausal state. Furthermore, as a primary function for the survival of the species is effective reproduction, it is likely that most of the advantages of having such post-menopausal risks relate to reproduction and the ability to address environmental stresses. This opinion/perspective will be discussed in the context of how such post-menopausal risks could enhance reproduction, with improved survival of offspring, and perhaps why such risks are preserved. Not all post-menopausal females exhibit risk for this set of diseases, and those who do develop such diseases do not have all of the conditions. The diseases of the post-menopausal state do not operate as a unified complex, but as independent variables, with the potential for some overlap. The how and why there would be such heterogeneity if the risk factors serve essential functions during the reproductive years is also discussed and the concept of sets of reversible epigenetic changes associated with puberty, pregnancy, and lactation is offered to explain the observations regarding the distribution of post-menopausal conditions and their potential roles in reproduction. While the involvement of an epigenetic system with a dynamic "modification-demodification-remodification" paradigm contributing to disease risk is a hypothesis at this point, validation of it could lead to a better understanding of post-menopausal disease risk in the context of reproduction with commonalities may also lead to future improved interventions to control such risk after menopause.

Extracellular Vesicles Generated by Mesenchymal Stem Cells in Stirred Suspension Bioreactors Promote Angiogenesis in Human-Brain-Derived Endothelial Cells.

Interrupted blood flow in the brain due to ischemic injuries such as ischemic stroke or traumatic brain injury results in irreversible brain damage, leading to cognitive impairment associated with inflammation, disruption of the blood-brain barrier (BBB), and cell death. Since the BBB only allows entry to a small class of drugs, many drugs used to treat ischemia in other tissues have failed in brain-related disorders. The administration of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) has shown promise in improving the functional recovery of the brain following cerebral ischemia by inducing blood vessel formation. To facilitate such a treatment approach, it is necessary to develop bioprocesses that can produce therapeutically relevant MSC-EVs in a reproducible and scalable manner. This study evaluated the feasibility of using stirred suspension bioreactors (SSBs) to scale-up the serum-free production of pro-angiogenic MSC-EVs under clinically relevant physioxic conditions. It was found that MSCs grown in SSBs generated EVs that stimulated angiogenesis in cerebral microvascular endothelial cells, supporting the use of SSBs to produce MSC-EVs for application in cerebral ischemia. These properties were impaired at higher cell confluency, outlining the importance of considering the time of harvest when developing bioprocesses to manufacture EV populations.

Complement factor D as a predictor of Achilles tendon healing and long-term patient outcomes.

Dense connective tissue healing, such as tendon, is protracted leading to highly variable and unsatisfactory patient outcomes. Biomarkers prognostic of long-term clinical outcomes is, however, unknown. The present study was designed to investigate the proteomic profile of healing, identify potential biomarkers, and assess their association with the patient's long-term outcomes after ATR. Quantitative mass spectrometry analysis demonstrated 1423 proteins in healing and contralateral healthy Achilles tendons of 28 ATR patients. Comparing healing at 2 weeks and healthy protein profiles, we identified 821 overlapping, 390 upregulated, and 17 downregulated proteins. Upregulated proteins are related mainly to extracellular matrix organization and metabolism, while downregulated pathways were associated with exocytosis in immune modulation and thrombosis formation. Further proteomic profiling in relation to validated patient outcomes revealed the downregulated pro-inflammatory complement factor D (CFD) as the most reliable predictive biomarker of successful tendon healing. Our finding showed a comprehensive proteomic landscape and bioinformatics on human connective tissue, indicating subtype-specific and shared biological processes and proteins in healing and healthy Achilles tendons, as well as in tendons related to good and poor patient outcomes. Inflammatory protein CFD and serpin family B member 1 were finally identified as potential predictive biomarkers of effective healing outcomes when combined the proteomic profiles with a validated clinical database. Following the future elucidation of the mechanisms associated with the identified biomarkers as predictors of good outcomes, our findings could lead to improved prognostic accuracy and development of targeted treatments, thus improving the long-term healing outcomes for all patients.

Use of Brain-Derived Stem/Progenitor Cells and Derived Extracellular Vesicles to Repair Damaged Neural Tissues: Lessons Learned from Connective Tissue Repair Regarding Variables Limiting Progress and Approaches to Overcome Limitations.

Pluripotent neural stem or progenitor cells (NSC/NPC) have been reported in the brains of adult preclinical models for decades, as have mesenchymal stem/stromal cells (MSC) been reported in a variety of tissues from adults. Based on their in vitro capabilities, these cell types have been used extensively in attempts to repair/regenerate brain and connective tissues, respectively. In addition, MSC have also been used in attempts to repair compromised brain centres. However, success in treating chronic neural degenerative conditions such as Alzheimer's disease, Parkinson's disease, and others with NSC/NPC has been limited, as have the use of MSC in the treatment of chronic osteoarthritis, a condition affecting millions of individuals. However, connective tissues are likely less complex than neural tissues regarding cell organization and regulatory integration, but some insights have been gleaned from the studies regarding connective tissue healing with MSC that may inform studies attempting to initiate repair and regeneration of neural tissues compromised acutely or chronically by trauma or disease. This review will discuss the similarities and differences in the applications of NSC/NPC and MSC, where some lessons have been learned, and potential approaches that could be used going forward to enhance progress in the application of cellular therapy to facilitate repair and regeneration of complex structures in the brain. In particular, variables that may need to be controlled to enhance success are discussed, as are different approaches such as the use of extracellular vesicles from stem/progenitor cells that could be used to stimulate endogenous cells to repair the tissues rather than consider cell replacement as the primary option. Caveats to all these efforts relate to whether cellular repair initiatives will have long-term success if the initiators for neural diseases are not controlled, and whether such cellular initiatives will have long-term success in a subset of patients if the neural diseases are heterogeneous and have multiple etiologies.

Development of a Porcine Model to Assess the Effect of In Situ Knee Joint Loading on Site-Specific Cartilage Gene Expression.

Cyclic mechanical loading of cartilage induces stresses and fluid flow, which are thought to modulate chondrocyte metabolism. The uneven surface, plus the heterogeneity of cartilage within a joint, makes stress and fluid pressure distribution in the tissue nonuniform, and gene expression may vary at different sites as a function of load magnitude, frequency, and time. In previous studies, cartilage explants were used for loading tests to investigate biological responses of the cartilage to mechanical loading. In contrast, we used loading tests on intact knee joints, to better reflect the loading conditions in a joint, and thus provide a more physiologically relevant mechanical environment. Gene expression levels in loaded samples for a selection of relevant genes were compared with those of the corresponding unloaded control samples to characterize potential differences. Furthermore, the effects of load magnitude and duration on gene expression levels were investigated. We observed differences in gene expression levels between samples from different sites in the same joint and between corresponding samples from the same site in loaded and unloaded joints. Consistent with previous findings, our results indicate that there is a critical upper and lower threshold of loading for triggering the expression of certain genes. Variations in gene expression levels may reflect the effect of local loading, topography, and structure of the cartilage in an intact joint on the metabolic activity of the associated cells.

One of the Primary Functions of Tissue-Resident Pluripotent Pericytes Cells May Be to Regulate Normal Organ Growth and Maturation: Implications for Attempts to Repair Tissues Later in Life.

Adult mesenchymal stem cells were reported more than 30 years ago. Since then, their potential to repair and regenerate damaged or diseased tissues has been studied intensively in both preclinical models and human trials. Most of the need for such tissue repair/regeneration is in older populations, so much of the effort has been performed with autologous cells in older patients. However, success has been difficult to achieve. In the literature, it has been noted that such progenitor cells from younger individuals often behave with more vigorous activity and are functionally enhanced compared to those from older individuals or animals. In addition, cells with the characteristics of mesenchymal stem cells or pluripotent mesenchymal regulatory cells exist in nearly all tissues and organs as pericytes since fetal life. Such evidence raises the possibility that one of the primary roles of these organ-specific cells is to regulate organ growth and maturation, and then subsequently play a role in the maintenance of organ integrity. This review will discuss the evidence to support this concept and the implications of such a concept regarding the use of these progenitor cells for the repair and regeneration of tissues damaged by injury or disease later in life. For the latter, it may be necessary to return the organ-specific progenitor cells to the functional state that contributed to their effectiveness during growth and maturation rather than attempting to use them after alterations imposed during the aging process have been established and their function compromised.

Sex Differences in Biological Systems and the Conundrum of Menopause: Potential Commonalities in Post-Menopausal Disease Mechanisms.

Sex-specific differences in biology and physiology likely start at the time of conception and progress and mature during the pre-puberty time frame and then during the transitions accompanying puberty. These sex differences are impacted by both genetics and epigenetic alterations during the maturation process, likely for the purpose of preparing for successful reproduction. For females, later in life (~45-50) they undergo another transition leading to a loss of ovarian hormone production at menopause. The reasons for menopause are not clear, but for a subset of females, menopause is accompanied by an increased risk of a number of diseases or conditions that impact a variety of tissues. Most research has mainly focused on the target cells in each of the affected tissues rather than pursue the alternative option that there may be commonalities in the development of these post-menopausal conditions in addition to influences on specific target cells. This review will address some of the potential commonalities presented by an integration of the literature regarding tissue-specific aspects of these post-menopausal conditions and data presented by space flight/microgravity (a condition not anticipated by evolution) that could implicate a loss of a regulatory function of the microvasculature in the risk attached to the affected tissues. Thus, the loss of the integration of the paracrine relationships between endothelial cells of the microvasculature of the tissues affected in the post-menopausal environment could contribute to the risk for post-menopausal diseases/conditions. The validation of this concept could lead to new approaches for interventions to treat post-menopausal conditions, as well as provide new understanding regarding sex-specific biological regulation.

Osteoarthritis as an Umbrella Term for Different Subsets of Humans Undergoing Joint Degeneration: The Need to Address the Differences to Develop Effective Conservative Treatments and Prevention Strategies.

Osteoarthritis (OA) of joints such as the knee and hip are very prevalent, and the number of individuals affected is expected to continue to rise. Currently, conservative treatments after OA diagnosis consist of a series of increasingly invasive interventions as the degeneration and pain increase, leading very often to joint replacement surgery. Most interventions are focused on alleviating pain, and there are no interventions currently available that stop and reverse OA-associated joint damage. For many decades OA was considered a disease of cartilage, but it is now considered a disease of the whole multi-tissue joint. As pain is the usual presenting symptom, for most patients, it is not known when the disease process was initiated and what the basis was for the initiation. The exception is post-traumatic OA which results from an overt injury to the joint that elevates the risk for OA development. This scenario leads to very long wait lists for joint replacement surgery in many jurisdictions. One aspect of why progress has been so slow in addressing the needs of patients is that OA has been used as an umbrella term that does not recognize that joint degeneration may arise from a variety of mechanistic causes that likely need separate analysis to identify interventions unique to each subtype (post-traumatic, metabolic, post-menopausal, growth and maturation associated). A second aspect of the slow pace of progress is that the bulk of research in the area is focused on post-traumatic OA (PTOA) in preclinical models that likely are not clearly relevant to human OA. That is, only ~12% of human OA is due to PTOA, but the bulk of studies investigate PTOA in rodents. Thus, much of the research community is failing the patient population affected by OA. A third aspect is that conservative treatment platforms are not specific to each OA subset, nor are they integrated into a coherent fashion for most patients. This review will discuss the literature relevant to the issues mentioned above and propose some of the directions that will be required going forward to enhance the impact of the research enterprise to affect patient outcomes.

Homo sapiens May Incorporate Daily Acute Cycles of "Conditioning-Deconditioning" to Maintain Musculoskeletal Integrity: Need to Integrate with Biological Clocks and Circadian Rhythm Mediators.

Human evolution required adaptation to the boundary conditions of Earth, including 1 g gravity. The bipedal mobility of Homo sapiens in that gravitational field causes ground reaction force (GRF) loading of their lower extremities, influencing the integrity of the tissues of those extremities. However, humans usually experience such loading during the day and then a period of relative unloading at night. Many studies have indicated that loading of tissues and cells of the musculoskeletal (MSK) system can inhibit their responses to biological mediators such as cytokines and growth factors. Such findings raise the possibility that humans use such cycles of acute conditioning and deconditioning of the cells and tissues of the MSK system to elaborate critical mediators and responsiveness in parallel with these cycles, particularly involving GRF loading. However, humans also experience circadian rhythms with the levels of a number of mediators influenced by day/night cycles, as well as various levels of biological clocks. Thus, if responsiveness to MSK-generated mediators also occurs during the unloaded part of the daily cycle, that response must be integrated with circadian variations as well. Furthermore, it is also possible that responsiveness to circadian rhythm mediators may be regulated by MSK tissue loading. This review will examine evidence for the above scenario and postulate how interactions could be both regulated and studied, and how extension of the acute cycles biased towards deconditioning could lead to loss of tissue integrity.

Methylprednisolone acetate mitigates IL1ß induced changes in matrix metalloproteinase gene expression in skeletally immature ovine explant knee tissues.

OBJECTIVE AND DESIGN: This study aimed at evaluating the effect of methylprednisolone (MPA) on messenger ribonucleic acid (mRNA) expression levels in immature ovine knee joint tissue explants following interleukin (IL)1ß induction and to assess responsiveness of the explants. MATERIAL OR SUBJECTS: Explants were harvested from the articular cartilage, synovium, and infrapatellar fat pad (IPFP) from immature female sheep. TREATMENT: Methylprednisolone. METHODS: The samples were allocated into six groups: (1) control, (2) MPA (10-3 M), (3) MPA (10-4 M), (4) IL1ß, (5) IL1ß + 10-3 M MPA, or (6) IL1ß + 10-4 M MPA. mRNA expression levels for molecules relevant to inflammation, cartilage degradation/anabolism, activation of innate immunity, and adipose tissue/hormones were quantified. Fold changes with MPA treatment were compared via the comparative CT method. RESULTS: Methylprednisolone treatment significantly suppressed MMPs consistently across the cartilage (MMP1, MMP3, and MMP13), synovium (MMP1 and MMP3), and IPFP (MMP13) (all p < 0.05). Other genes that were less consistently suppressed include endogenous IL1ß (cartilage) and IL6 (IPFP) (all p < 0.05), and others not affected either by IL-1 exposure or subsequent MPA include TGFß1, TLR4, and adipose-related molecules. CONCLUSIONS: Methylprednisolone significantly mitigated IL1ß induced mRNA expression for MMPs in the immature cartilage, synovium, and IPFP, but the extent of the responsiveness was tissue-, location-, and gene-specific.

What Molecular Recognition Systems Do Mesenchymal Stem Cells/Medicinal Signaling Cells (MSC) Use to Facilitate Cell-Cell and Cell Matrix Interactions? A Review of Evidence and Options.

Mesenchymal stem cells, also called medicinal signaling cells (MSC), have been studied regarding their potential to facilitate tissue repair for >30 years. Such cells, derived from multiple tissues and species, are capable of differentiation to a number of lineages (chondrocytes, adipocytes, bone cells). However, MSC are believed to be quite heterogeneous with regard to several characteristics, and the large number of studies performed thus far have met with limited or restricted success. Thus, there is more to understand about these cells, including the molecular recognition systems that are used by these cells to perform their functions, to enhance the realization of their potential to effect tissue repair. This perspective article reviews what is known regarding the recognition systems available to MSC, the possible systems that could be looked for, and alternatives to enhance their localization to specific injury sites and increase their subsequent facilitation of tissue repair. MSC are reported to express recognition molecules of the integrin family. However, there are a number of other recognition molecules that also could be involved such as lectins, inducible lectins, or even a MSC-specific family of molecules unique to these cells. Finally, it may be possible to engineer expression of recognition molecules on the surface of MSC to enhance their function in vivo artificially. Thus, improved understanding of recognition molecules on MSC could further their success in fostering tissue repair.

Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.

A maternal high-fat/sucrose diet, in the presence of maternal obesity, can program increased susceptibility to obesity and metabolic disease in offspring. In particular, nonalcoholic fatty liver disease risk is associated with poor maternal nutrition and obesity status, which may manifest via alterations in gut microbiota. Here, we report that in a preclinical model of diet-induced maternal obesity, maternal supplementation of a high-fat/sucrose diet with the prebiotic oligofructose improves glucose tolerance, insulin sensitivity, and hepatic steatosis in offspring following a long-term high-fat/sucrose dietary challenge compared with offspring of untreated dams. These improvements are associated with alterations in gut microbial composition and serum inflammatory profiles in early life and improvements in inflammatory and fatty-acid gene expression profiles in tissues. Serum metabolomics analysis highlights potential metabolic links between the gut microbiota and the degree of steatosis, including alterations in 1-carbon metabolism. Overall, our data suggest that maternal prebiotic intake protects offspring against hepatic steatosis and insulin resistance following 21 wk of high fat/sucrose diet, which is in part due to alterations in gut microbiota.-Paul, H. A., Collins, K. H., Nicolucci, A. C., Urbanski, S. J., Hart, D. A., Vogel, H. J., Reimer, R. A. Maternal prebiotic supplementation reduces fatty liver development in offspring through altered microbial and metabolomic profiles in rats.

Activation of NF-κB in Synovium versus Cartilage from Patients with Advanced Knee Osteoarthritis: A Potential Contributor to Inflammatory Aspects of Disease Progression.

The aim was to assess the activation and association of the NF-κB system across synovial membrane (SM) and articular cartilage (AC) in patients with knee osteoarthritis (OA) and ascertain its potential effects on catabolic mediator expression in advanced OA. SM and AC were obtained from 40 OA patients undergoing total knee arthroplasty and from 19 postmortem control subjects. NF-κB subunit RelA in nuclear and cytosolic fractions and NF-κB1-DNA binding in nuclear extracts was assessed by ELISA, whereas NFKB1, RELA, IL-8, IL-6, and MMP3 gene expression were analyzed by reverse transcriptase-quantitative PCR in tissues. We observed higher SM nuclear RelA protein levels and upregulated NF-κB1-DNA binding in OA patients compared with postmortem controls. However, in AC, lower nuclear RelA levels were observed compared with cytosolic extracts in patients. Nuclear RelA levels correlated positively with NF-κB1-DNA binding in SM and AC in patients. SM RELA and MMP3 mRNA levels were upregulated, whereas IL-8 and IL-6 as well as AC RELA were downregulated in patients compared with controls. In SM, nuclear RelA levels correlated positively with MMP3 gene expression in patients. A negative correlation was observed between SM nuclear RelA levels and AC NF-κB1-DNA binding, and SM nuclear NF-κB1-DNA binding correlated negatively with AC MMP3 and NFKB1 mRNA levels in patients. These findings highlight NF-κB-triggered cross-talk and feedback mechanisms between SM and AC in OA. Further, our findings strongly support a role for an activated NF-κB system in the transcriptional mechanism of inflammatory processes, especially in SM of patients with advanced OA.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts.

Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

NF-κB-Associated Pain-Related Neuropeptide Expression in Patients with Degenerative Disc Disease.

The role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) has been highlighted in mechanisms underlying inflammatory and neuropathic pain processes. The present study was designed to investigate whether NF-κB signaling is associated with pain-related neuropeptide expression in patients with chronic back pain related to degenerative disc disease (DDD). Intervertebral disc (IVD) tissues were collected from forty DDD patients undergoing disc replacement or fusion surgery, and from eighteen postmortem (PM) control subjects. RELA, NFKB1, CGRP, TAC1, TRPV1, and MMP-3 gene expression were analyzed by RT-qPCR, while NF-κB subunit RelA and NF-κB1⁻DNA binding in nuclear extracts and calcitonin gene related peptide (CGRP), substance P (SP), and transient receptor potential, subfamily V, member 1 (TRPV1) protein levels in cytosolic extracts of tissues were assessed by enzyme-linked immunosorbent assay (ELISA). An upregulated NF-κB1⁻DNA binding, and higher CGRP and TRPV1 protein levels were observed in DDD patients compared to PM controls. In DDD patients, NF-κB1⁻DNA binding was positively correlated with nuclear RelA levels. Moreover, NF-κB1⁻DNA binding was positively associated with TRPV1 and MMP-3 gene and SP and TRPV1 protein expression in DDD patients. Our results indicate that the expression of SP and TRPV1 in IVD tissues was associated with NF-κB activation. Moreover, NF-κB may be involved in the generation or maintenance of peripheral pain mechanisms by the regulation of pain-related neuropeptide expression in DDD patients.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts.

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-ß signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.