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Background: Despite its unique regenerative capacity, bone healing can be compromised, leading to delayed fracture regeneration and consequently nonunion. Due to the scarcity of autografts and the problems associated with a supraphysiological use of rhBMP-2, novel tissue engineering strategies arise as a promising solution to overcome nonunions and related bone pathologies. Purpose: To clinically deal with fracture nonunion, we designed engineered mimetic autografts consisting of a personalized polycaprolactone (PCL) scaffold surrounded by a porous PCL membrane mimicking the periosteum synthesized by melt electrowriting (MEW) (Figure 1). Methods: MEW membrane was functionalized with poly ethyl acrylate (PEA) and Fibronectin for efficient rhBMP-2 binding and delivery. The regenerative capacity and therapeutic potential of these scaffolds were tested in vitro for osteoblast differentiation and vivo in a critical size femur defect in Sprague Dawley rats (n=6-7 animals/group) (ethical approval 073-20). Regenerative effects were assessed by qPCR, q-mCT and histological analysis. Non-parametric Kruskal Wallis test was used for statistical analysis. Results: We selected the two lowest dose implants (10 mg/ml, 51.94±8.84 ng and 25 mg/ml, 186.8±17.33) to assess release profile over time and for in vivo therapeutic effect. In vitro, single loading of 186 ng of rhBMP-2 allows similar differentiation potential that standard osteogenic differentiation medium where fresh rhBMP-2 was added twice weekly (Figure 2). In vivo, regarding bone regeneration, quantitative μCT analysis shows great bone healing of defects treated with rhBMP-2 at concentrations of 25 μg/ml (186 ng) and 10 μg/ml (52 ng). Control group, 6.80±2.47 mm3; 10 μg/ml BMP-2 group 19.53±4.266 mm3, *p=0.0324; 25 μg/ml BMP-2 group 24.48±11.30 mm3, **p=0.0087. In addition, histological analysis was carried out to determine the osteoconductive potential of our PCL core (Figure 3). Conclusion: In conclusion, PEA functionalized mimetic periosteum show an unpreceded increase in bone healing, greatly enhancing rhBMP-2 effects.

Introduction: Despite its regenerative capacity, bone healing can be compromised, leading to delayed fracture regeneration and nonunion. Due to the scarcity of bone tissue that can be used as autograft, novel tissue engineering strategies arise as a promising solution by using biocompatible materials. Methods: Our objective is the development of engineered autografts capable of efficiently treat fracture nonunion. For this purpose, we designed polycaprolactone (PCL) autografts surrounded by a porous membrane mimicking periosteum. To assess their regenerative capacity, these scaffolds were tested in critical size femur defect for ten weeks carrying out μCT and histological analysis. Additionally, we are focusing on the generation of PCL biocomposites, such as poly ethyl-acrylate (PEA) covered PCL membranes which can enhance morphogen functionalization, reducing the effective BMP dose. Results: At the mCT level, structural mimetic PCL scaffolds, showed no significant difference in bone healing (Empty group, 11.47±4.93 mm3; MA, 14.95±3.09 mm3, p=0.1711). Histological analysis demonstrates that MEW PCL mimicking periosteum enhances bone growth, but insufficient for successful healing. However, once functionalized with PEA and BMP-2, these implants showed highly improved regeneration (CTL group, 11,47±4,93 mm3; BMP-2 group, 49,24±13,20 mm3, p = 0.0001). Figure 1. These implants were loaded with BMP-2 solutions previously studied in vitro to estimate morphogen dose, which resulted in 55.64±14.83 ng (n=6). Conclusions and discussion: In conclusion, PEA functionalized mimetic autografts show an important increase in bone healing, enhancing BMP-2 effects, which provide representative regeneration with a 100 folds lower dose than typically described in literature.

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).