The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application.

It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells (MSCs). However, the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflammatory response of MSCs remains unclear. This study aimed to investigate the effect of mechanical loading on the inflammatory response of MSCs seeded on scaffolds. Cyclic mechanical loading was applied to biofabricate the cell-scaffold composite for 15 min/day over 7, 14, or 21 days. At the predetermined time points, culture supernatant was collected for inflammatory mediator detection, and gene expression was analyzed by qRT-PCR. The results showed that the expression of inflammatory mediators (IL1B and IL8) was downregulated (p < 0.05) and the expression of ALP (p < 0.01) and COL1A1 (p < 0.05) was upregulated under mechanical loading. The cell-scaffold composites biofabricated with or without mechanical loading were freeze-dried to prepare extracellular matrix-based scaffolds (ECM-based scaffolds). Murine macrophages were seeded on the ECM-based scaffolds to evaluate their polarization. The ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying enhanced the expression of M2 polarization-related biomarkers (Arginase 1 and Mrc1, p < 0.05) of macrophages in vitro and increased bone volume/total volume ratio in vivo. Overall, these findings demonstrated that mechanical loading could dually modulate the inflammatory responses and osteogenic differentiation of MSCs. Besides, the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying facilitated the M2 polarization of macrophages in vitro and bone regeneration in vivo. Mechanical loading may be a promising biofabrication strategy for bone biomaterials.

The objective of this study was to investigate whether intermittent administration of parathyroid hormone [1–34] (PTH[1–34]) promotes tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in vivo. A rat model of ACL reconstruction with autograft was established at the left hind leg. Every day, injections of 60 μg PTH[1–34]/kg subcutaneously were given to the PTH group rats (n = 10) for four weeks, and the controls (n = 10) received saline. The tendon-bone healing process was evaluated by micro-CT, biomechanical test, histological and immunohistochemical analyses. The effects of PTH[1–34] on serum chemistry, bone microarchitecture and expression of the PTH receptor (PTH1R) and osteocalcin were determined. Administration of PTH[1–34] significantly increased serum levels of calcium, alkaline phosphatase (AP), osteocalcin and tartrate-resistant acid phosphatase (TRAP). The expression of PTH1R on both osteocytes and chondrocyte-like cells at the tendon-bone interface was increased in the PTH group. PTH[1–34] also enhanced the thickness and microarchitecture of trabecular bone according to the micro-CT analysis. The results imply that systematically intermittent administration of PTH[1–34] promotes tendon-bone healing at an early stage via up-regulated PTH1R. This method may enable a new strategy for the promotion of tendon-bone healing after ACL reconstruction.

The osteogenic microenvironment of bone-repairing materials plays a key role in accelerating bone regeneration but remains incompletely defined, which significantly limits the application of such bioactive materials. Here, the transcriptional landscapes of different osteogenic microenvironments, including three-dimensional (3D) hydroxyapatite (HA) scaffolds and osteogenic medium (OM), for mesenchymal stromal cells (MSCs) in vitro were mapped at single-cell resolution. Our findings suggested that an osteogenic process reminiscent of endochondral ossification occurred in HA scaffolds through sequential activation of osteogenic-related signaling pathways, along with inflammation and angiogenesis, but inhibition of adipogenesis and fibrosis. Moreover, we revealed the mechanism during OM-mediated osteogenesis involves the ZBTB16 and WNT signaling pathways. Heterogeneity of MSCs was also demonstrated. In vitro ossification of LRRC75A⁺ MSCs was shown to have better utilization of WNT-related ossification process, and PCDH10⁺ MSCs with superiority in hydroxyapatite-related osteogenic process. These findings provided further understanding of the cellular activity modulated by OM conditions and HA scaffolds, providing new insights for the improvement of osteogenic biomaterials. This atlas provides a blueprint for research on MSC heterogeneity and the osteogenic microenvironment of HA scaffolds and a database reference for the application of bioactive materials for bone regeneration.