Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling

Subacromial Bursa: A Neglected Tissue Is Gaining More and More Attention in Clinical and Experimental Research

The subacromial bursa has long been demolded as friction-reducing tissue, which is often linked to shoulder pain and, therefore, partially removed during shoulder surgery. Currently, the discovery of the stem cell potential of resident bursa-derived cells shed a new light on the subacromial bursa. In the meanwhile, this neglected tissue is gaining more attention as to how it can augment the regenerative properties of adjacent tissues such as rotator cuff tendons. Specifically, the tight fibrovascular network, a high growth factor content, and the large progenitor potential of bursa-derived cells could complement the deficits that a nearby rotator cuff injury might experience due to the fact of its low endogenous regeneration potential. This review deals with the question of whether bursal inflammation is only a pain generator or could also be an initiator of healing. Furthermore, several experimental models highlight potential therapeutic targets to overcome bursal inflammation and, thus, pain. More evidence is needed to fully elucidate a direct interplay between subacromial bursa and rotator cuff tendons. Increasing attention to tendon repair will help to guide future research and answer open questions such that novel treatment strategies could harvest the subacromial bursa's potential to support healing of nearby rotator cuff injuries

Subacromial Bursa: A Neglected Tissue Is Gaining More and More Attention in Clinical and Experimental Research

The subacromial bursa has long been demolded as friction-reducing tissue, which is often linked to shoulder pain and, therefore, partially removed during shoulder surgery. Currently, the discovery of the stem cell potential of resident bursa-derived cells shed a new light on the subacromial bursa. In the meanwhile, this neglected tissue is gaining more attention as to how it can augment the regenerative properties of adjacent tissues such as rotator cuff tendons. Specifically, the tight fibrovascular network, a high growth factor content, and the large progenitor potential of bursa-derived cells could complement the deficits that a nearby rotator cuff injury might experience due to the fact of its low endogenous regeneration potential. This review deals with the question of whether bursal inflammation is only a pain generator or could also be an initiator of healing. Furthermore, several experimental models highlight potential therapeutic targets to overcome bursal inflammation and, thus, pain. More evidence is needed to fully elucidate a direct interplay between subacromial bursa and rotator cuff tendons. Increasing attention to tendon repair will help to guide future research and answer open questions such that novel treatment strategies could harvest the subacromial bursa’s potential to support healing of nearby rotator cuff injuries

Do Matrix Metalloproteases and Tissue Inhibitors of Metalloproteases in Tenocytes of the Rotator Cuff Differ with Varying Donor Characteristics?

An imbalance between matrix metalloproteases (MMPs) and the tissue inhibitors of metalloproteases (TIMPs) may have a negative impact on the healing of rotator cuff tears. The aim of the project was to assess a possible relationship between clinical and radiographic characteristics of patients such as the age, sex, as well as the degenerative status of the tendon and the MMPs and TIMPs in their tenocyte-like cells (TLCs). TLCs were isolated from ruptured supraspinatus tendons and quantitative Real-Time PCR and ELISA was performed to analyze the expression and secretion of MMPs and TIMPs. In the present study, MMPs, mostly gelatinases and collagenases such as MMP-2, -9 and -13 showed an increased expression and protein secretion in TLCs of donors with higher age or degenerative status of the tendon. Furthermore, the expression and secretion of TIMP-1, -2 and -3 was enhanced with age, muscle fatty infiltration and tear size. The interaction between MMPs and TIMPs is a complex process, since TIMPs are not only inhibitors, but also activators of MMPs. This study shows that MMPs and TIMPs might play an important role in degenerative tendon pathologies

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2–4 days), middle (5–6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process. View Full-Tex

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers

an In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP- BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration

Bursa-Derived cells show a distinct mechano-response to physiological and pathological loading in vitro

The mechano-response of highly loaded tissues such as bones or tendons is well investigated, but knowledge regarding the mechano-responsiveness of adjacent tissues such as the subacromial bursa is missing. For a better understanding of the physiological role of the bursa as a friction-reducing structure in the joint, the study aimed to analyze whether and how bursa-derived cells respond to physiological and pathological mechanical loading. This might help to overcome some of the controversies in the field regarding the role of the bursa in the development and healing of shoulder pathologies. Cells of six donors seeded on collagen-coated silicon dishes were stimulated over 3 days for 1 or 4 h with 1, 5, or 10% strain. Orientation of the actin cytoskeleton, YAP nuclear translocation, and activation of non-muscle myosin II (NMM-II) were evaluated for 4 h stimulations to get a deeper insight into mechano-transduction processes. To investigate the potential of bursa-derived cells to adapt their matrix formation and remodeling according to mechanical loading, outcome measures included cell viability, gene expression of extracellular matrix and remodeling markers, and protein secretions. The orientation angle of the actin cytoskeleton increased toward a more perpendicular direction with increased loading and lowest variations for the 5% loading group. With 10% tension load, cells were visibly stressed, indicated by loss in actin density and slightly reduced cell viability. A significantly increased YAP nuclear translocation occurred for the 1% loading group with a similar trend for the 5% group. NMM-II activation was weak for all stimulation conditions. On the gene expression level, only the expression of TIMP2 was down-regulated in the 1 h group compared to control. On the protein level, collagen type I and MMP2 increased with higher/longer straining, respectively, whereas TIMP1 secretion was reduced, resulting in an MMP/TIMP imbalance. In conclusion, this study documents for the first time a clear mechano-responsiveness in bursa-derived cells with activation of mechano-transduction pathways and thus hint to a physiological function of mechanical loading in bursa-derived cells. This study represents the basis for further investigations, which might lead to improved treatment options of subacromial bursa-related pathologies in the future