Compositional mapping of the mature anterior cruciate ligament‐to‐bone insertion

The anterior cruciate ligament (ACL)‐to‐bone interface constitutes a complex, multi‐tissue structure comprised of contiguous ligament, non‐mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL‐to‐bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non‐mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513–2523, 2017.

     

Effect of bone morphogenetic protein 2 on tendon‐to‐bone healing in a canine flexor tendon model

Tendon‐to‐bone healing is typically poor, with a high rate of repair‐site rupture. Bone loss after tendon‐to‐bone repair may contribute to poor outcomes. Therefore, we hypothesized that the local application of the osteogenic growth factor bone morphogenetic protein 2 (BMP‐2) would promote bone formation, leading to improved repair‐site mechanical properties. Intrasynovial canine flexor tendons were injured in Zone 1 and repaired into bone tunnels in the distal phalanx. BMP‐2 was delivered to the repair site using either a calcium phosphate matrix (CPM) or a collagen sponge (COL) carrier. Each animal also received carrier alone in an adjacent repair to serve as an internal control. Repairs were evaluated at 21 days using biomechanical, radiographic, and histologic assays. Although an increase in osteoid formation was noted histologically, no significant increases in bone mineral density occurred. When excluding functional failures (i.e., ruptured and gapped repairs), mechanical properties were not different when comparing BMP‐2/CPM groups with carrier controls. A significantly higher percentage of BMP‐2 treated specimens had a maximum force <20 N compared to carrier controls. While tendon‐to‐bone healing can be enhanced by addressing the bone loss that typically occurs after surgical repair, the delivery of BMP‐2 using the concentrations and methods of the current study did not improve mechanical properties over carrier alone. The anticipated anabolic effect of BMP‐2 was insufficient in the short time frame of this study to counter the post‐repair loss of bone. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1702–1709, 2012     

Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo

Osteocytes are the most abundant cells in bone yet are the most challenging to study because they are embedded in a mineralized matrix. We generated a clonal cell line called IDG‐SW3 (for Immortomouse/Dmp1‐GFP‐SW3) from long‐bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T antigen regulated by interferon γ (IFN‐γ). Cells from these mice can be expanded at 33 °C in the presence of IFN‐γ and then allowed to resume their original phenotype at 37 °C in the absence of IFN‐γ. IDG‐SW3 cells are Dmp1‐GFP^? and T antigen⁺ under immortalizing conditions but Dmp1‐GFP⁺ and T antigen^? under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type 1 collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and fibroblast growth factor 23 (FGF‐23), regulated by parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D₃. When cultured on 3D matrices, they express Dmp1‐GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast‐to‐osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF‐23. © 2011 American Society for Bone and Mineral Research     

Sustained delivery of transforming growth factor beta three enhances tendon‐to‐bone healing in a rat model

Despite advances in surgical technique, rotator cuff repairs are plagued by a high rate of failure. This failure rate is in part due to poor tendon‐to‐bone healing; rather than regeneration of a fibrocartilaginous attachment, the repair is filled with disorganized fibrovascular (scar) tissue. Transforming growth factor beta 3 (TGF‐β3) has been implicated in fetal development and scarless fetal healing and, thus, exogenous addition of TGF‐β3 may enhance tendon‐to‐bone healing. We hypothesized that: TGF‐β3 could be released in a controlled manner using a heparin/fibrin‐based delivery system (HBDS); and delivery of TGF‐β3 at the healing tendon‐to‐bone insertion would lead to improvements in biomechanical properties compared to untreated controls. After demonstrating that the release kinetics of TGF‐β3 could be controlled using a HBDS in vitro, matrices were incorporated at the repaired supraspinatus tendon‐to‐bone insertions of rats. Animals were sacrificed at 14–56 days. Repaired insertions were assessed using histology (for inflammation, vascularity, and cell proliferation) and biomechanics (for structural and mechanical properties). TGF‐β3 treatment in vivo accelerated the healing process, with increases in inflammation, cellularity, vascularity, and cell proliferation at the early timepoints. Moreover, sustained delivery of TGF‐β3 to the healing tendon‐to‐bone insertion led to significant improvements in structural properties at 28 days and in material properties at 56 days compared to controls. We concluded that TGF‐β3 delivered at a sustained rate using a HBDS enhanced tendon‐to‐bone healing in a rat model. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1099–1105, 2011     

Extracorporeal shockwave enhanced regeneration of fibrocartilage in a delayed tendon‐bone insertion repair model

Fibrous tissue is often formed in delayed healing of tendon bone insertion (TBI) instead of fibrocartilage. Extracorporeal shockwave (ESW) provides mechanical cues and upregulates expression of fibrocartilage‐related makers and cytokines. We hypothesized that ESW would accelerate fibrocartilage regeneration at the healing interface in a delayed TBI healing model. Partial patellectomy with shielding at the TBI interface was performed on 32 female New Zealand White Rabbits for establishing this delayed TBI healing model. The rabbits were separated into the control and ESW group for evaluations at postoperative week 8 and 12. Shielding was removed at week 4 and a single ESW treatment was applied at week 6. Fibrocartilage regeneration was evaluated histomorphologically and immunohistochemically. Vickers hardness of the TBI matrix was measured by micro‐indentation. ESW group showed higher fibrocartilage area, thickness, and proteoglycan deposition than the control in week 8 and 12. ESW increased expression of SOX9 and collagen II significantly in week 8 and 12, respectively. ESW group showed a gradual transition of hardness from bone to fibrocartilage to tendon, and had a higher Vickers hardness than the control group at week 12. In conclusion, ESW enhanced fibrocartilage regeneration at the healing interface in a delayed TBI healing model. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:507–514, 2014.     

Development of the supraspinatus tendon‐to‐bone insertion: Localized expression of extracellular matrix and growth factor genes

The adult healing response of the rotator cuff tendon‐to‐bone insertion site differs from the ordered process of insertion site development. Healing is characterized by disorganized scar and a lack of fibrocartilage formation, in contrast to the well organized fibrocartilaginous transition which forms during the normal development of the tendon‐to‐bone insertion. The purpose of this study was to localize the expression of a number of extracellular matrix and growth factor genes during insertion site development in order to guide future strategies for augmenting adult rotator cuff healing. The rotator cuff was morphologically distinct at 13.5 dpc (days postconception). Neo‐tendon was evident as a condensation of cells adjacent to bone. The interface between tendon and bone did not form into a mature fibrocartilaginous insertion until 21‐days postnatally, based upon the appearance of four distinct zones with a mineralized humeral head. Fibroblasts of the supraspinatus tendon expressed type I collagen at all timepoints. Type II collagen was first expressed by chondrocytes in the fibrocartilage and mineralized fibrocartilage at 7 days and persisted in the mineralized fibrocartilage at 56 days. Type X collagen was first expressed by the chondrocytes in the mineralized fibrocartilage at 14 days and persisted in the mineralized fibrocartilage at 56 days. A shift from TGF‐β3 to TGF‐β1 expression occurred at 15.5 dpc. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1621–1628, 2007     

Role of myeloid early endothelial progenitor cells in bone formation and osteoclast differentiation in tissue construct based on hydroxyapatite poly(ester‐urethane) scaffolds

Engineering of a vascularized bone construct is a highly challenging task which needs to take into account the impact of different components on the bone regeneration process. Bone repair influencing factors in such constructs range from the material properties and scaffold design, to the interaction of different cell types contributing to bone formation and remodeling or neovascularization, respectively. In this context, early endothelial progenitor cells (EPC), mononuclear cells isolated from the peripheral blood, express the endothelial marker CD31 but also a series of myeloid markers and have been shown to support the formation of vessel‐like structures. These cells are also characterized by a highly adaptable phenotype influenced by other cells creating an instructive niche. The present study was designed to investigate the impact of EPC on bone formation or remodeling using a co‐culture system of outgrowth endothelial cells, mature endothelial cells isolated from the peripheral blood cell cultures, and mesenchymal stem cells grown on hydroxyapatite poly(ester‐urethane) scaffolds. The formation of vessel‐like structures in these constructs was shown by CLSM and immunohistochemistry and further evaluated by real time RT‐PCR. Osteogenic differentiation in these constructs was investigated by von Kossa, Alizarin Red, and real time PCR. Data indicated that osteogenic differentiation occurred within the constructs after 14 days of culture but without a direct influence by EPC in this process. Finally, although we observed a series of osteoclast related makers in the constructs when EPC were included, no indications for an increased osteoclast‐like activity, which might lead to increased bone resorption, were observed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1922–1932, 2016.     

The effect of muscle loading on flexor tendon‐to‐bone healing in a canine model

Previous tendon and ligament studies have demonstrated a role for mechanical loading in tissue homeostasis and healing. In uninjured musculoskeletal tissues, increased loading leads to an increase in mechanical properties, whereas decreased loading leads to a decrease in mechanical properties. The role of loading on healing tissues is less clear. We studied tendon‐to‐bone healing in a canine flexor tendon‐to‐bone injury and repair model. To examine the effect of muscle loading on tendon‐to‐bone healing, repaired tendons were either cut proximally (unloaded group) to remove all load from the distal phalanx repair site or left intact proximally (loaded group). All paws were casted postoperatively and subjected to daily passive motion rehabilitation. Specimens were tested to determine functional properties, biomechanical properties, repair‐site gapping, and bone mineral density. Loading across the repair site led to improved functional and biomechanical properties (e.g., stiffness for the loaded group was 8.2 ± 3.9 versus 5.1 ± 2.5 N/mm for the unloaded group). Loading did not affect bone mineral density or gapping. The formation of a gap between the healing tendon and bone correlated with failure properties. Using a clinically relevant model of flexor tendon injury and repair, we found that muscle loading was beneficial to healing. Complete removal of load by proximal transection resulted in tendon‐to‐bone repairs with less range of motion and lower biomechanical properties compared to repairs in which the muscle‐tendon‐bone unit was left intact. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

ACL reconstruction using bone‐tendon‐bone graft engineered from the semitendinosus tendon by injection of recombinant BMP‐2 in a rabbit model

We attempted to generate a bone‐tendon‐bone structure by injecting human‐type recombinant human bone morphogenetic protein‐2 (rhBMP‐2) into the semitendinosus tendon, and an anterior cruciate ligament (ACL) defect was reconstructed by grafting the engineered bone‐tendon‐bone graft. Two ossicles with a separation distance of 1 cm were generated within the left semitendinosus tendon of a rabbit 6 weeks after the injection of rhBMP‐2 (15 µg at each site). The engineered bone‐tendon‐bone graft was transplanted in order to reconstruct the ACL by passing the graft through the bone tunnels. In the control group, the ACL was reconstructed with the semitendinosus tendon without BMP‐2 using the same methods as those used in the experimental group. The animals were harvested at 4 or 8 weeks after surgery and examined by radiographic, histological, and biomechanical methods. In the experimental group, ossicles in the bone‐tendon‐bone graft were successfully integrated into the host bone of the femur and tibia. Histological analysis revealed that characteristic features identical to the normal direct insertion morphology had been restored. Biomechanical pull‐out testing showed that the ultimate failure load and stiffness of the reconstructed ACL in the experimental group were significantly higher than those in the control group at both 4 and 8 weeks (p < 0.05). These results indicate the potential of regenerative reconstruction of the ACL, and the reconstruction resulted in the restoration of morphology and function equivalent to those of the normal ACL. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1923–1930, 2011     

Experimental micromechanics of the cement–bone interface

Despite the widespread use of cement as a means of fixation of implants to bone, surprisingly little is known about the micromechanical behavior in terms of the local interfacial motion. In this work, we utilized digital image correlation techniques to quantify the micromechanics of the cement–bone interface of laboratory‐prepared cemented total hip replacements subjected to nondestructive, quasistatic tensile and compressive loading. Upon loading, the majority of the displacement response localized at the contact interface region between cement and bone. The contact interface was more compliant (p = 0.0001) in tension (0.0067 ± 0.0039 mm/MPa) than compression (0.0051 ± 0.0031 mm/MPa), and substantial hysteresis occurred due to sliding contact between cement and bone. The tensile strength of the cement–bone interface was inversely proportional to the compliance of the interface and proportional to the cement/bone contact area. When loaded beyond the ultimate strength, the strain localization process continued at the contact interface between cement and bone with microcracking (damage) to both. More overall damage occurred to the cement than to the bone. The opening and closing at the contact interface from loading could serve as a conduit for submicron size particles. In addition, the cement mantle is not mechanically supported by surrounding bone as optimally as is commonly assumed. Both effects may influence the longevity of the reconstruction and could be considered in preclinical tests. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:872–879, 2008     

Local injection of thrombin‐related peptide (TP508) in PPF/PLGA microparticles–enhanced bone formation during distraction osteogenesis

We have previously demonstrated that injections of the thrombin‐related peptide, TP508, into the lengthening gap have significantly enhanced bone consolidation in a rabbit model of distraction osteogenesis. This study was to further test the effect of a single TP508 injection in slow release preparation on bone formation during distraction osteogenesis. Rabbits had left tibiae lengthened unilateral lengthener at rate of 1.4 mm/day for 6 days. TP508 was injected into as the following: Group 1, TP508 in saline; Group 2, in PPF/PLGA [poly(propylene fumarate)/poly(D,L ‐lactic‐co‐glycolic acid)] microparticles; and Group 3, dextran gel only. All the animals were killed 2 weeks after lengthening. On radiographies, more bone was formed in the two TP508‐treated groups at first and secnd week postlengthening than that of the control Group 3. Microcomputed tomography (microCT) at 2 weeks indicated that the most advanced bone formation and remodeling was seen in Group 2. The mean volumetric BMD of the regenerates was significantly higher in the TP508 treated groups compared to the control group (p < 0.05). Histological evaluations supported the radiographic and the microCT results. In conclusion, we have demonstrated that a single injection of small amount of TP508 (300 µg) at the end of lengthening phases has significantly enhanced bone consolidation process in a rabbit model of distraction osteogenesis. The delivery of TP508 in PPF/PLGA microparticles appears to lead to a better quality bone formation over the saline delivery, further examinations are needed to confirm if PPF/PLGA microparticles may be desirable drug delivery form in augmenting bone formation. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:539–546, 2008     

Interindividual anatomical variations affect the plate‐to‐bone fit during osteosynthesis of distal radius fractures

We hypothesized that interindividual variations in the teardrop, which represents the volar projection of the lunate facet of the distal radius, cause unsatisfactory fitting of the volar locking plate to the bone. This can cause flexor tendon ruptures. Herein, we conducted a cross‐sectional study and measured the ratio of teardrop height and the teardrop inclination angle as parameters of teardrop configuration for 200 standardized lateral radiographs (average age of the patients, 51 years). We also quantified the influence of the teardrop morphology by analyzing the fit of three locking plates to three radii with differing teardrop inclination angles using a three‐dimensional computer‐aided design system. The average ratios of the teardrop height and teardrop inclination angle were 0.42° (0.30–0.56°) and 28.8° (9.9–44.9°), respectively. The teardrop inclination angle was moderately correlated with age in men but not in women. In the plate‐to‐bone fit analyses, the fit of all the plates was significantly different between bones, with the configuration of the radius with the lowest teardrop inclination angle being the closest approximation to that of each plate. We demonstrated the interindividual variation in the shape of the teardrop and its influence on the fit of the volar plate, highlighting the importance of careful plate selection for achieving osteosynthesis of bones with a high teardrop inclination angle. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:953–960, 2016.     

Micromechanics of postmortem‐retrieved cement–bone interfaces

The cement–bone interface plays an important role in load transfer between cemented implant systems and adjacent bone, but little is known about the micromechanical behavior of this interface following in vivo service. Small samples of postmortem‐retrieved cement–bone specimens from cemented total hip replacements were prepared and mechanically loaded to determine the response to tensile and compressive loading. The morphology of the cement–bone interface was quantified using a CT‐based stereology approach. Laboratory‐prepared specimens were used to represent immediate postoperative conditions for comparison. The stiffness and strength of the cement–bone interface from postmortem retrievals was much lower than that measured from laboratory‐prepared specimens. The cement–bone interfaces from postmortem retrievals were very compliant (under tension and compression) and had a very low tensile strength (0.21 ± 0.32 MPa). A linear regression model, including interface contact fraction and intersection fraction between cement and bone, could explain 71% (p < 0.0001) of the variability in experimental response. Bony remodeling following an arthroplasty procedure may contribute to reduced contact between cement and bone, resulting in weaker, more compliant interfaces. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:170–177, 2010     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011     

Influence of bone adaptation on tendon‐to‐bone healing in bone tunnel after anterior cruciate ligament reconstruction in a rabbit model

Anterior cruciate ligament (ACL) reconstruction with placement of grafted tendon in bone tunnel is a common surgical procedure. Bone tunnel creation may result in stress shielding of postero‐lateral regions of tibial tunnel. The present study was designed to characterize the changes of peri‐graft bone and compare with tendon‐to‐bone (T‐B) healing in spatial and temporal manners after ACL reconstruction in rabbit. Surgical reconstruction using digital extensor tendon in bone tunnel was performed on 48 rabbits. Twelve rabbits were sacrificed at 0, 2, 6, and 12 weeks postoperatively for radiological and histological examinations. Bone mass and microarchitecture at the anterior, posterior, medial, and lateral regions of tunnel wall at distal femur and proximal tibia were evaluated. Using peripheral quantitative computed tomography, a 26, 22, and 42% decrease in bone mineral density (BMD) relative to baseline was present in the medial region of the femoral tunnel and the posterior and lateral regions of the tibial tunnel, respectively, at week 12 postoperatively (p < 0.05). It was accompanied by a decrease in trabecular number and increase in trabecular spacing, the shift of platelike to rodlike trabeculae, and loss of anisotropy under micro‐computed tomography evaluation. This finding was echoed by histology showing increased osteoclastic activities and poor T‐B healing in these regions. In conclusion, the postoperative bone loss and associated poor T‐B healing was region‐dependent, which may result from adaptive changes after tunnel creation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1447–1456, 2009