A finite element inverse analysis to assess functional improvement during the fracture healing process

Assessment of the restoration of load-bearing function is the central goal in the study of fracture healing process. During the fracture healing, two critical aspects affect its analysis: (1) material properties of the callus components, and (2) the spatio-temporal architecture of the callus with respect to cartilage and new bone formation. In this study, an inverse problem methodology is used which takes into account both features and yields material property estimates that can analyze the healing changes. Six stabilized fractured mouse tibias are obtained at two time points during the most active phase of the healing process, respectively 10 days (n=3), and 14 days (n=3) after fracture. Under the same displacement conditions, the inverse procedure estimations of the callus material properties are generated and compared to other fracture healing metrics. The FEA estimated property is the only metric shown to be statistically significant (p=0.0194) in detecting the changes in the stiffness that occur during the healing time points. In addition, simulation studies regarding sensitivity to initial guess and noise are presented; as well as the influence of callus architecture on the FEA estimated material property metric. The finite element model inverse analysis developed can be used to determine the effects of genetics or therapeutic manipulations on fracture healing in rodents

Goodpasture Antigen-binding Protein Is a Soluble Exportable Protein That Interacts with Type IV Collagen

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at leasttwoGPBPisoforms resultingfromcanonical (77- kDa) and noncanonical (91-kDa)mRNAtranslation initiation.The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. Theseandprevious data implicateGPBPsin a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.This work was supported by Grants SAF97/0065, SAF2000/0047, SAF2001/0453, SAF2003-09772-C03-01, and SAF2006-12520-C02-01 from Ministerio de Educacio´ n y Ciencia, Grant 98/102-00 from Fundacio ´ n “La Caixa,” and Grants GV04B-285 and BM-001/2002 from Generalitat Valenciana (Spain) (to J. S.).Medicin

Comparison of microCT and an inverse finite element approach for biomechanical analysis: Results in a MSC therapeutic system for fracture healing

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures

Mechanical barriers and transforming growth factor beta inhibitor on epidural fibrosis in a rabbit laminectomy model

Background: TGF-β has been described as a mediator of fibrosis and scarring. Several studies achieved reduction in experimental scarring through the inhibition of TGF-β. Fibroblasts have been defined as the cell population originating fibrosis, blocking fibroblast invasion may impair epidural fibrosis appearance. For this purpose, biocompatible materials used as mechanical barriers and a TGF-β inhibitor peptide were evaluated in the reduction of epidural fibrosis. Methods: A L6 laminectomy was performed in 40 New Zealand white rabbits. Divided into four groups, each rabbit was assigned to receive either collagen sponge scaffold (CS group), gelatin-based gel (GCP group), P144® (iTGFβ group), or left untreated (control group). Four weeks after surgery, cell density, collagen content, and new bone formation of the scar area were determined by histomorphometry. Two experienced pathologists scored dura mater adhesion, scar density, and inflammatory infiltrate in a blinded manner. Results: In all groups, laminectomy site was filled with fibrous tissue and the dura mater presented adhesions. Only GCP group presented a significant reduction in collagen content and scar density. Conclusion: GCP treatment reduces epidural fibrosis although did not prevent dura mater adhesion completely

Mesenchymal Stem Cells Expressing Insulin-like Growth Factor-I (MSCIGF) Promote Fracture Healing and Restore New Bone Formation in Irs1 Knockout Mice: Analyses of MSCIGF Autocrine and Paracrine Regenerative Effects

Failures of fracture repair (non-unions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe and feasible. The contributions of MSC to the reparative process can occur through autocrine as well as paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSC expressing insulin-like growth factor-I (MSCIGF), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and non-unions. MSCIGF or traceable MSCIGF-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knock-out (Irs1−/−) mice with a stabilized tibia fracture. Healing was assed using biomechanical testing, micro-computed-tomography (µCT) and histological analyses. We found that systemically transplanted MSCIGF through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSCIGF to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced induced osteoglastogenesis in MSC was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1−/− mice as a non-union fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap; a callus that basically absent in Irs1−/− left untransplanted or transplanted with MSC. We provided evidence of effects and mechanisms for transplanted MSCIGF in fracture repair and potentially to treat non-unions

CC-Chemokine Receptor-2 Expression in Osteoblasts Contributes to Cartilage and Bone Damage during Post-Traumatic Osteoarthritis

In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1α-expressing cells to obtain osteoblasts lacking Ccr2 (CCR2-Col1αKO). We stimulated PTOA changes in CCR2-Col1αKO and CCR2+/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast-Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2+/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1α-Ccr2 deletion led to less evident improvements. Osteoblast-Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues

Regenerative Effects of Transplanted Mesenchymal Stem Cells in Fracture Healing

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat non-unions. The use of MSC to treat fractures is attractive as it would be implementing a reparative process that should be in place but occurs to be defective or protracted and MSC effects would be needed only for the repairing time that is relatively brief. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed-tomography and histological studies. Transplanting CMV-Cre-R26R-LacZ-MSC, we found that MSC engrafted within the callus endosteal niche. Using MSC from BMP-2-Lac-Z mice genetically modified using a bacterial artificial chromosome system to be β-gal reporters for BMP-2 expression, we found that MSC contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow to design novel MSC-based therapies to treat fractures

Tissue engineered scaffolds for mimetic autografts

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

Tissue engineered mimetic periosteum for efficient delivery of rhBMP-2

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

Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice

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 invasio