Histological and immunohistochemical guide to tendon tissue.

Tendons are unique dense connective tissues with discrete zones having specific structure and function. They are juxtaposed with other tissues (e.g., bone, muscle, and fat) with different compositional, structural, and mechanical properties. Additionally, tendon properties change drastically with growth and development, disease, aging, and injury. Consequently, there are unique challenges to performing high quality histological assessment of this tissue. To address this need, histological assessment was one of the breakout session topics at the 2022 Orthopaedic Research Society (ORS) Tendon Conference hosted at the University of Pennsylvania. The purpose of the breakout session was to discuss needs from members of the ORS Tendon Section related to histological procedures, data presentation, knowledge dissemination, and guidelines for future work. Therefore, this review provides a brief overview of the outcomes of this discussion and provides a set of guidelines, based on the perspectives from our laboratories, for histological assessment to assist researchers in their quest to utilize these techniques to enhance the outcomes and interpretations of their studies.

Targeting the hedgehog signaling pathway to improve tendon-to-bone integration.

OBJECTIVE: While the role of hedgehog (Hh) signaling in promoting zonal fibrocartilage production during development is well-established, whether this pathway can be leveraged to improve tendon-to-bone repair in adults is unknown. Our objective was to genetically and pharmacologically stimulate the Hh pathway in cells that give rise to zonal fibrocartilaginous attachments to promote tendon-to-bone integration. DESIGN: Hh signaling was stimulated genetically via constitutive Smo (SmoM2 construct) activation of bone marrow stromal cells or pharmacologically via systemic agonist delivery to mice following anterior cruciate ligament reconstruction (ACLR). To assess tunnel integration, we measured mineralized fibrocartilage (MFC) formation in these mice 28 days post-surgery and performed tunnel pullout testing. RESULTS: Hh pathway-related genes increased in cells forming the zonal attachments in wild-type mice. Both genetic and pharmacologic stimulation of the Hh pathway increased MFC formation and integration strength 28 days post-surgery. We next conducted studies to define the role of Hh in specific stages of the tunnel integration process. We found Hh agonist treatment increased the proliferation of the progenitor pool in the first week post-surgery. Additionally, genetic stimulation led to continued MFC production in the later stages of the integration process. These results indicate that Hh signaling plays an important biphasic role in cell proliferation and differentiation towards fibrochondrocytes following ACLR. CONCLUSION: This study reveals a biphasic role for Hh signaling during the tendon-to-bone integration process after ACLR. In addition, the Hh pathway is a promising therapeutic target to improve tendon-to-bone repair outcomes.

Sustained delivery of vascular endothelial growth factor from mesoporous calcium-deficient hydroxyapatite microparticles promotes in vitro angiogenesis and osteogenesis.

Promoting the growth of blood vessels within engineered tissues remains one of the main challenge in bone tissue engineering. One way to improve angiogenesis is the use of vascular endothelial growth factor (VEGF) as it holds the ability to increase the formation of a vascular network. In the present study, collagen scaffolds with VEGF-releasing hydroxyapatite particles were fabricated, in order to engineer a material both capable of presenting an osteoconductive surface and delivering an angiogenic growth factor in a localized and sustained manner, in order to enhance osteogenesis as well as angiogenesis. To this end, we developed microparticles and characterize their size, chemical properties and Ca/P ratio to validate the formation of hydroxyapatite. We then evaluated the osteogenic potential of HAp when cultured with mesenchymal stem cells and compare it to commercially available hydroxyapatite (SBp). Finally, we characterized the encapsulation and release of VEGF in the HAp and assess the angiogenic potential of the VEGF-HAp when cultured with endothelial cells. We demonstrated the successful fabrication of calcium deficient hydroxyapatite microparticles (CDHAp), with biological properties closer to the bone than stoichiometric, commercially available hydroxyapatite. This CDHAp exhibited a well-defined 3D network of crystalline nanoplates forming mesoporous and hollow structures. The high specific area created by those structures enabled the loading of VEGF with high efficiency when compared to the loading efficiency of SBp. Furthermore, their biological performances were evaluated in vitro. Our results indicate that VEGF-CDHAp can be used to improve both osteogenesis and angiogenesis in vitro.

Amplifying Bone Marrow Progenitors Expressing α-Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon-to-Bone Repair.

Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.

Bioprinted osteon-like scaffolds enhance in vivo neovascularization.

Bone tissue engineers are facing a daunting challenge when attempting to fabricate bigger constructs intended for use in the treatment of large bone defects, which is the vascularization of the graft. Cell-based approaches and, in particular, the use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. We present in this paper an alternative method to mimic the spatial pattern of HUVECs and hMSCs found in native osteons based on the use of extrusion-based 3D bioprinting (3DP). We developed a 3DP biphasic osteon-like scaffold, containing two separate osteogenic and vasculogenic cell populations encapsulated in a fibrin bioink in order to improve neovascularization. To this end, we optimized the fibrin bioink to improve the resolution of printed strands and ensure a reproducible printing process; the influence of printing parameters on extruded strand diameter and cell survival was also investigated. The mechanical strength of the construct was improved by co-printing the fibrin bioink along a supporting PCL carrier scaffold. Compressive mechanical testing showed improved mechanical properties with an average compressive modulus of 131 ± 23 MPa, which falls in the range of cortical bone. HUVEC and hMSC laden fibrin hydrogels were printed in osteon-like patterns and cultured in vitro. A significant increase in gene expression of angiogenic markers was observed for the biomimetic scaffolds. Finally, biphasic scaffolds were implanted subcutaneously in rats. Histological analysis of explanted scaffolds showed a significant increase in the number of blood vessels per area in the 3D printed osteon-like scaffolds. The utilization of these scaffolds in constructing biomimetic osteons for bone regeneration demonstrated a promising capacity to improve neovascularization of the construct. These results indicates that proper cell orientation and scaffold design could play a critical role in neovascularization.

Fabrication and evaluation of 3D printed BCP scaffolds reinforced with ZrO2 for bone tissue applications.

Fused deposition modeling (FDM) is a promising 3D printing and manufacturing step to create well interconnected porous scaffold designs from the computer-aided design (CAD) models for the next generation of bone scaffolds. The purpose of this study was to fabricate and evaluate a new biphasic calcium phosphate (BCP) scaffold reinforced with zirconia (ZrO2 ) by a FDM system for bone tissue engineering. The 3D slurry foams with blending agents were successfully fabricated by a FDM system. Blending materials were then removed after the sintering process at high temperature to obtain a targeted BCP/ZrO2 scaffold with the desired pore characteristics, porosity, and dimension. Morphology of the sintered scaffold was investigated with SEM/EDS mapping. A cell proliferation test was carried out and evaluated with osteosarcoma MG-63 cells. Mechanical testing and cell proliferation evaluation demonstrated that 90% BCP and 10% ZrO2 scaffold had a significant effect on the mechanical properties maintaining a structure compared that of only 100% BCP with no ZrO2 . Additionally, differentiation studies of human mesenchymal stem cells (hMSCs) on BCP/ZrO2 scaffolds in static and dynamic culture conditions showed increased expression of bone morphogenic protein-2 (BMP-2) when cultured on BCP/ZrO2 scaffolds under dynamic conditions compared to on BCP control scaffolds. The manufacturing of BCP/ZrO2 scaffolds through this innovative technique of a FDM may provide applications for various types of tissue regeneration, including bone and cartilage.