Alayna E. Loiselle

Rescue of Impaired Fracture Healing in COX-2−/− Mice via Activation of Prostaglandin E2 Receptor Subtype 4

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2(-/-) mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2(-/-) mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2(-/-) callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2(-/-) mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2(-/-) fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2(-/-) mice.

Remodeling of Murine Intrasynovial Tendon Adhesions following Injury: MMP and Neotendon Gene Expression

Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real‐time RT‐PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp‐9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp‐2 and Mmp‐14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf‐5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.

Gap junction and hemichannel functions in osteocytes

Cell-to-cell and cell-to-matrix communication in bone cells mediated by gap junctions and hemichannels, respectively, maintains bone homeostasis. Gap junctional communication between cells permits the passage of small molecules including calcium and cyclic AMP. This cell-to-cell communication occurs between bone cells including osteoblasts, osteoclasts and osteocytes, and is important in both bone formation and bone resorption. Connexin (Cx) 43 is the predominant gap junction protein in bone cells, and facilitates the communication of cellular signals either through docking of gap junctions between two cells, or through the formation of un-paired hemichannels. Systemic deletion of Cx43 results in perinatal lethality, so conditional deletion models are necessary to study the postnatal role of gap junctions in bone. These models provide the opportunity to determine the role of gap junctions in specific bone cells, notably the osteocyte. In this review, we summarize the key roles that gap junctions and hemichannels in osteocytes play in bone cell response to many stimuli including mechanical loading, intracellular and extracellular stimuli, such as parathyroid hormone, PGE2, plasma calcium levels and pH, as well as in maintaining osteocyte survival.

Impact of Smad3 loss of function on scarring and adhesion formation during tendon healing.

Studies were performed evaluating the role of Smad3, a transcription factor mediating canonical TGF‐β signaling, on scarring and adhesion formation using an established flexor digitorum longus (FDL) tendon repair model. In unoperated animals the metatarsophalangeal (MTP) range of motion (ROM) was similar in Smad3−/− and wild‐type (WT) mice while the basal tensile strength of Smad3−/− tendons was significantly (39%) lower than in WT controls. At 14 and 21 days following repair Smad3−/− MTP ROM reached approximately 50% of the basal level and was twice that observed in WT tendon repairs, consistent with reduced adhesion formation. Smad3−/− and WT maximal tensile repair strength on post‐operative day 14 was similar. However, Smad3−/− tendon repairs maximal tensile strength on day 21 was 42% lower than observed in matched WT mice, mimicking the relative decrease in strength observed in Smad3−/− FDL tendons under basal conditions. Histology showed reduced “healing callus” in Smad3−/− tendons while quantitative PCR, in situ hybridization, and immunohistochemistry showed decreased col3a1 and col1a1 and increased MMP9 gene and protein expression in repaired Smad3−/− tendons. Thus, Smad3−/− mice have reduced collagen and increased MMP9 gene and protein expression and decreased scarring following tendon FDL tendon repair.

TAK1 Regulates Cartilage and Joint Development via the MAPK and BMP Signaling Pathways

The importance of canonical transforming growth factor β (TGF‐β) and bone morphogenetic protein (BMP) signaling during cartilage and joint development is well established, but the necessity for noncanonical (SMAD‐independent) signaling during these processes is largely unknown. TGF‐β activated kinase 1 (TAK1) is a MAP3K activated by TGF‐β, BMP, and other mitogen‐activated protein kinase (MAPK) signaling components. We set out to define the potential role for noncanonical, TAK1‐mediated signaling in cartilage and joint development via deletion of Tak1 in chondrocytes (Col2Cre;Tak1f/f) and the developing limb mesenchyme (Prx1Cre;Tak1f/f). Deletion of Tak1 in chondrocytes resulted in novel embryonic developmental cartilage defects including decreased chondrocyte proliferation, reduced proliferating chondrocyte survival, delayed onset of hypertrophy, reduced Mmp13 expression, and a failure to maintain interzone cells of the elbow joint, which were not observed previously in another Col2Cre;Tak1f/f model. Deletion of Tak1 in limb mesenchyme resulted in widespread joint fusions likely owing to the differentiation of interzone cells to the chondrocyte lineage. The Prx1Cre;Tak1f/f model also allowed us to identify novel columnar chondrocyte organization and terminal maturation defects owing to the interplay between chondrocytes and the surrounding mesenchyme. Furthermore, both our in vivo models and in vitro cell culture studies demonstrate that loss of Tak1 results in impaired activation of the downstream MAPK target p38, as well as diminished activation of the BMP/SMAD signaling pathway. Taken together, these data demonstrate that TAK1 is a critical regulator of both MAPK and BMP signaling and is necessary for proper cartilage and joint development.

Connexin 43 deficiency desensitizes bone to the effects of mechanical unloading through modulation of both arms of bone remodeling

Connexin 43 (Cx43) is a gap junction protein that plays an integral role in the skeletal response to mechanical loading and unloading. In a previous study, we demonstrated preservation of trabecular bone mass and cortical bone formation rate in mice with an osteoblast/osteocyte-selective deficiency of Cx43 (cKO) following mechanical unloading via hindlimb suspension (HLS). In the present study, we sought to define the potential mechanisms underlying this response. Following three weeks of HLS, mRNA levels of Sost were significantly greater in wild-type (WT)-Suspended mice vs. WT-Control, while there was no difference between cKO control and cKO-Suspended. Unloading-induced decreases in P1NP, a serum marker of bone formation, were also attenuated in cKO-Suspended. The proportion of sclerostin-positive osteocytes was significantly lower in cKO-Control vs. WT-Control (-72%, p<0.05), a difference accounted for by the presence of numerous empty lacunae in the cortical bone of cKO vs. WT. Abundant TUNEL staining was present throughout the cortical bone of the tibia and femur, suggesting an apoptotic process. There was no difference in empty lacunae in the trabecular bone of the tibia or femur. Trabecular and cortical osteoclast indices were lower in cKO-Suspended vs. WT-Suspended; however, mRNA levels of the gene encoding RANKL increased similarly in both genotypes. Connexin 43 deficient mice experience attenuated sclerostin-mediated suppression of cortical bone formation and lower cortical osteoclast activity during unloading. Preservation of trabecular bone mass and attenuated osteoclast activity during unloading, despite an apparent lack of effect on osteocyte viability at this site, suggests that an additional mechanism independent of osteocyte apoptosis may also be important. These findings indicate that Cx43 is able to modulate both arms of bone remodeling during unloading.

Optimizing the osteogenic potential of adult stem cells for skeletal regeneration

Adult stem cells, including mesenchymal stem cells, display plasticity in that they can differentiate toward various lineages including bone cells, cartilage cells, fat cells, and other types of connective tissue cells. However, it is not clear what factors direct adult stem cell lineage commitment and terminal differentiation. Emerging evidence suggests that extracellular physical cues have the potential to control stem cell lineage specification. In this perspective article, we review recent findings on biomaterial surface and mechanical signal regulation of stem cell differentiation. Specifically, we focus on stem cell response to substrate nanoscale topography and fluid flow induced shear stress and how these physical factors may regulate stem cell osteoblastic differentiation in vitro.

Osteoblast and osteocyte‐specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing

Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte‐specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin‐Cre+; Cx43flox/flox (Cx43cKO) and Cre‐; Cx43flox/flox (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte‐specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post‐fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing.

Shifting paradigms on the role of connexin43 in the skeletal response to mechanical load.

Gap junctions (GJs) are membrane‐spanning channels that allow for the movement of small molecules across cell membranes. Connexin43 (Cx43) is the predominant GJ protein in bone. In vitro studies suggest that gap junctional intercellular communication (GJIC) sensitizes bone cells to mechanical signals. Additionally, mechanical signals detected by osteocytes are communicated to osteoblasts via GJIC, and osteocytic Cx43 hemichannels release anabolic factors, such as PGE2 and ATP, in response to mechanical load. These findings and others have led to near consensus among researchers in the field that GJIC, hemichannels or connexins facilitate the anabolic response of bone to mechanical load and, in their absence, bone would be less sensitive to load. However, recent in vivo evidence suggests the opposite is true. Studies from our laboratory and others demonstrate that Cx43‐deficient mice have an increased anabolic response to mechanical load and are protected against the catabolic effects of mechanical unloading. These developments suggest a paradigm shift in our understanding of connexins, GJIC, and mechanotransduction in bone. That is, inhibiting bone cell Cx43 expression or GJIC has a beneficial effect on bones response to its mechanical environment, preserving bone during unloading and enhancing its formation during loading. Here, we review literature in support of this hypothesis and suggest a mechanism by which Cx43, through interaction with WNT/β‐catenin signaling, moderates both arms of bone remodeling.

Development of antisense oligonucleotide (ASO) technology against Tgf‐β signaling to prevent scarring during flexor tendon repair

Flexor tendons (FT) in the hand provide near frictionless gliding to facilitate hand function. Upon injury and surgical repair, satisfactory healing is hampered by fibrous adhesions between the tendon and synovial sheath. In the present study we used antisense oligonucleotides (ASOs), specifically targeted to components of Tgf‐β signaling, including Tgf‐β1, Smad3 and Ctgf, to test the hypothesis that local delivery of ASOs and suppression of Tgf‐β1 signaling would enhance murine FT healing by suppressing adhesion formation while maintaining strength. ASOs were injected in to the FT repair site at 2, 6 and 12 days post‐surgery. ASO treatment suppressed target gene expression through 21 days. Treatment with Tgf‐β1, Smad3 or Ctgf ASOs resulted in significant improvement in tendon gliding function at 14 and 21 days, relative to control. Consistent with a decrease in adhesions, Col3a1 expression was significantly decreased in Tgf‐β1, Smad3 and Ctgf ASO treated tendons relative to control. Smad3 ASO treatment enhanced the maximum load at failure of healing tendons at 14 days, relative to control. Taken together, these data support the use of ASO treatment to improve FT repair, and suggest that modulation of the Tgf‐β1 signaling pathway can reduce adhesions while maintaining the strength of the repair.