Torsion constants and virtual mechanical tests are valid image-based surrogate measures of ovine fracture healing.

In large animal studies, the mechanical reintegration of the bone fragments is measured using postmortem physical testing, but these assessments can only be performed once, after sacrifice. Image-based virtual mechanical testing is an attractive alternative because it could be used to monitor healing longitudinally. However, the procedures and software required to perform finite element analysis (FEA) on subject-specific models for virtual mechanical testing can be time consuming and costly. Accordingly, the goal of this study was to determine whether a simpler image-based geometric measure-the torsion constant, sometimes known as polar moment of inertia-can be reliably used as a surrogate measure of bone healing in large animals. To achieve this, postmortem biomechanical testing and microCT scans were analyzed for a total of 33 operated and 20 intact ovine tibiae. An image-processing procedure to compute the attenuation-weighted torsion constant from the microCT scans was developed in MATLAB and this code has been made freely available. Linear regression analysis was performed between the postmortem biomechanical data, the results of virtual mechanical testing using FEA, and the torsion constants measured from the scans. The results showed that virtual mechanical testing is the most reliable surrogate measure of postmortem torsional rigidity, having strong correlations and high absolute agreement. However, when FEA is not practical, the torsion constant is a viable alternative surrogate measure that is moderately correlated with postmortem torsional rigidity and can be readily calculated.

Studying Edema Formation After Release of the Infraspinatus Muscle as an Experimental Model of Rotator Cuff Lesions in Sheep: A Histological Analysis.

BACKGROUND: Muscle edema formation and inflammatory processes are early manifestations of acute rotator cuff lesions in sheep. Histological analysis of affected muscles revealed edema formation, inflammatory changes, and muscle tissue disruption in MRs. HYPOTHESIS: Edema contributes to inflammatory reactions and early muscle fiber degeneration before the onset of fatty infiltration. STUDY DESIGN: Controlled laboratory study. METHODS: Osteotomy of the greater tuberosity, including the insertion of the infraspinatus tendon, was performed on 14 sheep. These experimental animal models were divided into 2 groups: a nontrauma group with surgical muscle release alone (7 sheep) and a trauma group with standardized application of additional trauma to the musculotendinous unit (7 sheep). Excisional biopsy specimens of the infraspinatus muscle were taken at 0, 3, and 4 weeks. RESULTS: Edema formation was histologically demonstrated in both groups and peaked at 3 weeks. At 3 weeks, signs of muscle fiber degeneration were observed. At 4 weeks, ingrowth of loose alveolar and fibrotic tissue between fibers was detected. Fatty tissue was absent. The diameter of muscle fibers increased in both groups, albeit to a lesser degree in the trauma group, and practically normalized at 4 weeks. Immunohistology revealed an increase in macrophage types 1 and 2, as well as inflammatory mediators such as prostaglandin E2 and nuclear factor kappa-light-chain-enhancer of activated B cells. CONCLUSION: Early muscle edema and concomitant inflammation precede muscle fiber degeneration and fibrosis. Edema formation results from tendon release alone and is only slightly intensified by additional trauma. CLINICAL RELEVANCE: This study illustrates that early edema formation and inflammation elicit muscle fiber degeneration that precedes fatty infiltration. Should this phenomenon be applicable to human traumatic rotator cuff tears, then surgery should be performed as soon as possible, ideally within the first 21 days after injury.

Rotator Cuff Repair and Overlay Augmentation by Direct Interlocking of a Nonwoven Polyethylene Terephthalate Patch Into the Tendon: Evaluation in an Ovine Model.

BACKGROUND: Arthroscopic repair of large rotator cuff tendon tears is associated with high rates of retear. Construct failure often occurs at the suture-tendon interface. Patch augmentation can improve mechanical strength and healing at this interface. PURPOSE: To introduce a novel technique for suture-free attachment of an overlaid patch and evaluate its biomechanical strength and biological performance. STUDY DESIGN: Descriptive and controlled laboratory studies. METHODS: An established ovine model of partial infraspinatus tendon resection and immediate repair was used. After a nonwoven polyethylene terephthalate patch was overlaid to the resected tendon, a barbed microblade was used to draw fibers of the patch directly into the underlying tissue. In vivo histological assessment of healing was performed at 6 and 13 weeks after implantation. Ex vivo models were used to characterize primary repair strength of the suture-free patch fixation to tendon. Additional ex vivo testing assessed the potential of the technique for patch overlay augmentation of suture-based repair. RESULTS: The in vivo study revealed no macroscopic evidence of adverse tissue reactions to the interlocked patch fibers. Histological testing indicated a normal host healing response with minimal fibrosis. Uniform and aligned tissue ingrowth to the core of the patch was observed from both the tendon and the bone interfaces to the patch. There was no evident retraction of the infraspinatus muscle, lengthening of the tendon, or tendon gap formation over 13 weeks. Ex vivo testing revealed that direct patch interlocking yielded tendon purchase equivalent to a Mason-Allen suture (150 ± 58 vs 154 ± 49 N, respectively; P = .25). In an overlay configuration, fiber interlocked patch augmentation increased Mason-Allen suture retention strength by 88% (from 221 ± 43 N to 417 ± 86 N; P < .01) with no detectable difference in repair stiffness. CONCLUSION: Testing in an ovine model of rotator cuff tendon repair suggested that surgical interlocking of a nonwoven medical textile can provide effective biomechanical performance, support functional tissue ingrowth, and help avoid musculotendinous retraction after surgical tendon repair. CLINICAL RELEVANCE: The novel technique may facilitate patch augmentation of rotator cuff repairs.

Autologous and Allogeneic Cytotherapies for Large Knee (Osteo)Chondral Defects: Manufacturing Process Benchmarking and Parallel Functional Qualification.

Cytotherapies are often necessary for the management of symptomatic large knee (osteo)-chondral defects. While autologous chondrocyte implantation (ACI) has been clinically used for 30 years, allogeneic cells (clinical-grade FE002 primary chondroprogenitors) have been investigated in translational settings (Swiss progenitor cell transplantation program). The aim of this study was to comparatively assess autologous and allogeneic approaches (quality, safety, functional attributes) to cell-based knee chondrotherapies developed for clinical use. Protocol benchmarking from a manufacturing process and control viewpoint enabled us to highlight the respective advantages and risks. Safety data (telomerase and soft agarose colony formation assays, high passage cell senescence) and risk analyses were reported for the allogeneic FE002 cellular active substance in preparation for an autologous to allogeneic clinical protocol transposition. Validation results on autologous bioengineered grafts (autologous chondrocyte-bearing Chondro-Gide scaffolds) confirmed significant chondrogenic induction (COL2 and ACAN upregulation, extracellular matrix synthesis) after 2 weeks of co-culture. Allogeneic grafts (bearing FE002 primary chondroprogenitors) displayed comparable endpoint quality and functionality attributes. Parameters of translational relevance (transport medium, finished product suturability) were validated for the allogeneic protocol. Notably, the process-based benchmarking of both approaches highlighted the key advantages of allogeneic FE002 cell-bearing grafts (reduced cellular variability, enhanced process standardization, rationalized logistical and clinical pathways). Overall, this study built on our robust knowledge and local experience with ACI (long-term safety and efficacy), setting an appropriate standard for further clinical investigations into allogeneic progenitor cell-based orthopedic protocols.

Combined electric and magnetic field therapy for bone repair and regeneration: an investigation in a 3-mm and an augmented 17-mm tibia osteotomy model in sheep.

BACKGROUND: Therapies using electromagnetic field technology show evidence of enhanced bone regeneration at the fracture site, potentially preventing delayed or nonunions. METHODS: Combined electric and magnetic field (CEMF) treatment was evaluated in two standardized sheep tibia osteotomy models: a 3-mm non-critical size gap model and a 17-mm critical size defect model augmented with autologous bone grafts, both stabilized with locking compression plates. CEMF treatment was delivered across the fracture gap twice daily for 90 min, starting 4 days postoperatively (post-OP) until sacrifice (9 or 12 weeks post-OP, respectively). Control groups received no CEMF treatment. Bone healing was evaluated radiographically, morphometrically (micro-CT), biomechanically and histologically. RESULTS: In the 3-mm gap model, the CEMF group (n = 6) exhibited higher callus mineral density compared to the Control group (n = 6), two-fold higher biomechanical torsional rigidity and a histologically more advanced callus maturity (no statistically significant differences). In the 17-mm graft model, differences between the Control (n = 6) and CEMF group (n = 6) were more pronounced. The CEMF group showed a radiologically more advanced callus, a higher callus volume (p = 0.003) and a 2.6 × higher biomechanical torsional rigidity (p = 0.024), combined with a histologically more advanced callus maturity and healing. CONCLUSIONS: This study showed that CEMF therapy notably enhanced bone healing resulting in better new bone structure, callus morphology and superior biomechanical properties. This technology could transform a standard inert orthopedic implant into an active device stimulating bone tissue for accelerated healing and regeneration.

Histological Preparation and Evaluation of Cartilage Specimens.

In this chapter, an introduction is given into histological techniques to research related to hyaline cartilage and subchondral bone. Emphasis is placed on the importance to investigate cartilage and bone as a unit, which includes the transition zone of the calcified cartilage and tidemark. Reasons for the appropriate selection of histological methods are presented such as when to use (decalcified) specimens for routine paraffin embedding including immunohistology, cryosections of cartilage alone, or non-decalcified specimens for embedding in polymethylmethacrylate with or without additional biomaterials. Appropriate staining methods are also outlined. Apart from detailed laboratory protocols for different embedding and staining methods including open communication about difficulties related to the various techniques, also practical instructions for state-of-the-art evaluation methods and their strengths and weaknesses are given. Sample figures for scoring methods are included.

Studying Edema Formation After Release of the Infraspinatus Tendon as an Experimental Model of Rotator Cuff Tears in Sheep: A Preliminary Imaging and Morphological Analysis.

BACKGROUND: The cause, extent, and role of muscle edema for muscle degeneration are unknown and not considered in the current literature. In vivo experiments were designed to prove muscle edema formation in the early period in a sheep model of acute rotator cuff tears. HYPOTHESIS: Muscle edema occurs after tendon release with or without additional stretching trauma and may be associated with muscle retraction and subsequent muscle degeneration. STUDY DESIGN: Controlled laboratory study. METHODS: A sheep model with acute release of the infraspinatus tendon was used. An osteotomy of the greater tuberosity, including the insertion of the infraspinatus tendon, was performed in 14 sheep. To demonstrate presence of edema, magnetic resonance imaging scans were performed at 0, 2, and 4 weeks using T1-weighted, T2-weighted, proton density-weighted, and Dixon sequences. Excisional biopsy specimens were taken at 0, 3, and 4 weeks (histological results will be reported in a later publication). Two injury models were created: a nontrauma group that consisted of muscle release alone and a trauma group that included additional standardized traction to the musculotendinous unit. Evaluation of T1- and T2-weighted images included calculation of pennation angle, muscle fiber length, signal intensity (edema), and muscle volume. Muscle wet weight and volume were measured at sacrifice. RESULTS: Edema formation was shown in all sheep and slightly more pronounced in the trauma group, where muscle intensity increased significantly between time point 0 (200 Grey Value (GV)) and weeks 2, 3, and 4 (300 GV). Edema formation started early after tendon release with a plateau between 3 and 4 weeks. Deterioration of muscle fiber bundles began also after tendon release with a peak at 4 weeks. Muscle volume decreased steadily over time. CONCLUSION: Muscle edema appeared early after rotator cuff tendon release, was more pronounced in the trauma group, and reached a plateau after 3 to 4 weeks. Muscle fatty content decreased within the short period of 4 weeks owing to a dilution effect. Muscle edema seems to be an essential factor in cuff tears and subsequent muscle retraction and degeneration. CLINICAL RELEVANCE: This study demonstrates a new type of muscle edema of retraction and describes the characteristics of edema associated with a retracted rotator cuff tear.

Biomechanical duality of fracture healing captured using virtual mechanical testing and validated in ovine bones.

Bone fractures commonly repair by forming a bridging structure called callus, which begins as soft tissue and gradually ossifies to restore rigidity to the bone. Virtual mechanical testing is a promising technique for image-based assessment of structural bone healing in both preclinical and clinical settings, but its accuracy depends on the validity of the material model used to assign tissue mechanical properties. The goal of this study was to develop a constitutive model for callus that captures the heterogeneity and biomechanical duality of the callus, which contains both soft tissue and woven bone. To achieve this, a large-scale optimization analysis was performed on 2363 variations of 3D finite element models derived from computed tomography (CT) scans of 33 osteotomized sheep under normal and delayed healing conditions. A piecewise material model was identified that produced high absolute agreement between virtual and physical tests by differentiating between soft and hard callus based on radiodensity. The results showed that the structural integrity of a healing long bone is conferred by an internal architecture of mineralized hard callus that is supported by interstitial soft tissue. These findings suggest that with appropriate material modeling, virtual mechanical testing is a reliable surrogate for physical biomechanical testing.

Image-based radiodensity profilometry measures early remodeling at the bone-callus interface in sheep.

Bone healing has been traditionally described as a four-phase process: inflammatory response, soft callus formation, hard callus development, and remodeling. The remodeling phase has been largely neglected in most numerical mechanoregulation models of fracture repair in favor of capturing early healing using a pre-defined callus domain. However, in vivo evidence suggests that remodeling occurs concurrently with repair and causes changes in cortical bone adjacent to callus that are typically neglected in numerical models of bone healing. The objective of this study was to use image processing techniques to quantify this early-stage remodeling in ovine osteotomies. To accomplish this, we developed a numerical method for radiodensity profilometry with optimization-based curve fitting to mathematically model the bone density gradients in the radial direction across the cortical wall and callus. After assessing data from 26 sheep, we defined a dimensionless density fitting function that revealed significant remodeling occurring in the cortical wall adjacent to callus during early healing, a 23% average reduction in density compared to intact. This fitting function is robust for modeling radial density gradients in both intact bone and fracture repair scenarios and can capture a wide variety of the healing responses. The fitting function can also be scaled easily for comparison to numerical model predictions and may be useful for validating future mechanoregulatory models of coupled fracture repair and remodeling.

Optimizing large-scale autologous human keratinocyte sheets for major burns-Toward an animal-free production and a more accessible clinical application.

BACKGROUND AND AIMS: Autologous keratinocyte sheets constitute an important component of the burn wound treatment toolbox available to a surgeon and can be considered a life-saving procedure for patients with severe burns over 50% of their total body surface area. Large-scale keratinocyte sheet cultivation still fundamentally relies on the use of animal components such as inactivated murine 3T3 fibroblasts as feeders, animal-derived enzymes such as trypsin, as well as media components such as fetal bovine serum (FBS). This study was therefore aimed to optimize autologous keratinocyte sheets by comparing various alternatives to critical components in their production. METHODS: Human skin samples were retrieved from remnant operative tissues. Cell isolation efficiency and viability were investigated by comparing the efficacy of porcine-derived trypsin and animal-free enzymes (Accutase and TrypLESelect). The subsequent expansion of the cells and the keratinocyte sheet formation was analyzed, comparing various cell culture substrates (inactivated murine 3T3 fibroblasts, inactivated human fibroblasts, Collagen I or plain tissue culture plastic), as well as media containing serum or chemically defined animal-free media. RESULTS: The cell isolation step showed clear cell yield advantages when using porcine-derived trypsin, compared to animal-free alternatives. The keratinocyte sheets produced using animal-free serum were similar to those produced using 3T3 feeder layer and FBS-containing medium, particularly in mechanical integrity as all grafts were liftable. In addition, sheets grown on collagen in an animal-free medium showed indications of advantages in homogeneity, speed, reduced variability, and differentiation status compared to the other growth conditions investigated. Most importantly, the procedure was compatible with the up-scaling requirements of major burn wound treatments. CONCLUSION: This study demonstrated that animal-free components could be used successfully to reduce the risk profile of large-scale autologous keratinocyte sheet production, and thereby increase clinical accessibility.

Engineered nasal cartilage for the repair of osteoarthritic knee cartilage defects.

Osteoarthritis (OA) is the most prevalent joint disorder, causing pain and disability predominantly in the aging population but also affecting young individuals. Current treatments are limited to use of anti-inflammatory drugs to alleviate symptoms or degenerated joint replacement by a prosthetic implant at the end stage of the disease. We hypothesized that degenerative cartilage defects can be treated using nasal chondrocyte­based tissue-engineered cartilage (N-TEC). We demonstrate that N-TEC maintained cartilaginous properties when exposed in vitro to inflammatory stimuli found in osteoarthritic joints and favorably altered the inflammatory profile of cells from osteoarthritic joints. These effects were at least partially mediated by down-regulation of the WNT (wingless/integrated) signaling pathway through sFRP1 (secreted frizzled-related protein-1). We further report that N-TEC survive and engraft in vivo in ectopic mouse models reproducing a human osteochondral OA tissue environment, as well as in sheep articular cartilage defects that mimic degenerative settings. Last, we tested the safety of autologous N-TEC for the treatment of osteoarthritic cartilage defects in the knees of two patients with advanced OA (Kellgren and Lawrence grades 3 and 4) who were otherwise considered for unicondylar knee arthroplasty. No adverse reactions were recorded, and patients reported reduced pain as well as improved joint function and life quality 14 months after surgery. Together, our findings indicate that N-TEC can directly contribute to cartilage repair in osteoarthritic joints. A suitably powered clinical trial is now required to assess its efficacy in the treatment of patients with OA.

Optical fibers for endoscopic high-power Er:YAG laserosteotomy.

SIGNIFICANCE: The highest absorption peaks of the main components of bone are in the mid-infrared region, making Er:YAG and CO2 lasers the most efficient lasers for cutting bone. Yet, studies of deep bone ablation in minimally invasive settings are very limited, as finding suitable materials for coupling high-power laser light with low attenuation beyond 2 µm is not trivial. AIM: The first aim of this study was to compare the performance of different optical fibers in terms of transmitting Er:YAG laser light with a 2.94-µm wavelength at high pulse energy close to 1 J. The second aim was to achieve deep bone ablation using the best-performing fiber, as determined by our experiments. APPROACH: In our study, various optical fibers with low attenuation (λ = 2.94 µm) were used to couple the Er:YAG laser. The fibers were made of germanium oxide, sapphire, zirconium fluoride, and hollow-core silica, respectively. We compared the fibers in terms of transmission efficiency, resistance to high Er:YAG laser energy, and bending flexibility. The best-performing fiber was used to achieve deep bone ablation in a minimally invasive setting. To do this, we adapted the optimal settings for free-space deep bone ablation with an Er:YAG laser found in a previous study. RESULTS: Three of the fibers endured energy per pulse as high as 820 mJ at a repetition rate of 10 Hz. The best-performing fiber, made of germanium oxide, provided higher transmission efficiency and greater bending flexibility than the other fibers. With an output energy of 370 mJ per pulse at 10 Hz repetition rate, we reached a cutting depth of 6.82 ± 0.99 mm in sheep bone. Histology image analysis was performed on the bone tissue adjacent to the laser ablation crater; the images did not show any structural damage. CONCLUSIONS: The findings suggest that our prototype could be used in future generations of endoscopic devices for minimally invasive laserosteotomy.

Establishment of a localized acute implant-associated Staphylococcus aureus bone infection model in sheep.

Orthopedic implant-associated bacterial infections with Staphylococcus aureus constitute a major clinical problem, and large pre-clinical animal models remain scarce. The aim of this study was to establish a standardized method of a localized, acute S. aureus bone infection in the presence of complex implanted devices in a sheep model. Four sheep underwent surgery receiving a complex implanted metallic device with a component stabilizing a bone defect created in the left tibial metaphysis, and an attached component placed in adjacent soft tissue. The bone defect was inoculated with S. aureus strain ATCC25293 (1 × 104 CFU). Twenty one days later, the surgery site was macroscopically evaluated, tissue samples and implants harvested for bacterial cell count quantification and tissue samples histologically analyzed. The animals exhibited clinical signs of localized infection (e.g. swelling, lameness, pain) but did not develop symptoms of sepsis. After euthanasia, macroscopic assessment revealed a localized bone and soft tissue infection at the surgery site. Histologically, an acute inflammation with neutrophils but also signs of bone destruction with necrosis was noted. An ovine model of a localized, acute S. aureus bone infection with complex implants was successfully established and could be used to test novel treatments against orthopedic implant-associated infections.

Industrial Development of Standardized Fetal Progenitor Cell Therapy for Tendon Regenerative Medicine: Preliminary Safety in Xenogeneic Transplantation.

Tendon defects require multimodal therapeutic management over extensive periods and incur high collateral burden with frequent functional losses. Specific cell therapies have recently been developed in parallel to surgical techniques for managing acute and degenerative tendon tissue affections, to optimally stimulate resurgence of structure and function. Cultured primary human fetal progenitor tenocytes (hFPT) have been preliminarily considered for allogeneic homologous cell therapies, and have been characterized as stable, consistent, and sustainable cell sources in vitro. Herein, optimized therapeutic cell sourcing from a single organ donation, industrial transposition of multi-tiered progenitor cell banking, and preliminary preclinical safety of an established hFPT cell source (i.e., FE002-Ten cell type) were investigated. Results underlined high robustness of FE002-Ten hFPTs and suitability for sustainable manufacturing upscaling within optimized biobanking workflows. Absence of toxicity or tumorigenicity of hFPTs was demonstrated in ovo and in vitro, respectively. Furthermore, a 6-week pilot good laboratory practice (GLP) safety study using a rabbit patellar tendon partial-thickness defect model preliminarily confirmed preclinical safety of hFPT-based standardized transplants, wherein no immune reactions, product rejection, or tumour formation were observed. Such results strengthen the rationale of the multimodal Swiss fetal progenitor cell transplantation program and prompt further investigation around such cell sources in preclinical and clinical settings for musculoskeletal regenerative medicine.

Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment.

BACKGROUND AIMS: Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes. METHODS: Keratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development. RESULTS: The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors. CONCLUSIONS: These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients.

Domain-independent simulation of physiologically relevant callus shape in mechanoregulated models of fracture healing.

Mechanoregulatory models have been used to predict the progression of bone fracture healing for more than two decades. However, many published studies share the same fundamental limitation: callus development proceeds within a pre-defined domain that both restricts and directs healing and leads to some non-physiologic healing patterns. To address this limitation, we added two spatial proximity functions to an existing mechanoregulatory model of fracture healing to control the localization of callus within the healing domain. We tested the performance of the new model in an idealized ovine tibial osteotomy with medial plate fixation using three sizes of healing domains and multiple variations of the spatial proximity functions. All model variations produced outward callus growth and bridging weighted toward the far cortex, which is consistent with in vivo healing. With and without the proximity functions, there were marked differences in the predicted callus volume and shape. With no proximity functions, the callus produced was strongly domain dependent, with a 15% difference in volume between the smallest and largest initialization domains. With proximity function control, callus growth was restricted to near the fracture line and there was only 2% difference in volume between domain sizes. Superimposing both proximity functions - one to control outward growth and one representing a decay in periosteal activity away from the fracture - produced a predicted callus size that was within the physiologic range for sheep and had a realistic morphology when compared with fluorescent dye co-localization with calcium deposition over time and histology.

Development of Standardized Fetal Progenitor Cell Therapy for Cartilage Regenerative Medicine: Industrial Transposition and Preliminary Safety in Xenogeneic Transplantation.

Diverse cell therapy approaches constitute prime developmental prospects for managing acute or degenerative cartilaginous tissue affections, synergistically complementing specific surgical solutions. Bone marrow stimulation (i.e., microfracture) remains a standard technique for cartilage repair promotion, despite incurring the adverse generation of fibrocartilagenous scar tissue, while matrix-induced autologous chondrocyte implantation (MACI) and alternative autologous cell-based approaches may partly circumvent this effect. Autologous chondrocytes remain standard cell sources, yet arrays of alternative therapeutic biologicals present great potential for regenerative medicine. Cultured human epiphyseal chondro-progenitors (hECP) were proposed as sustainable, safe, and stable candidates for chaperoning cartilage repair or regeneration. This study describes the development and industrial transposition of hECP multi-tiered cell banking following a single organ donation, as well as preliminary preclinical hECP safety. Optimized cell banking workflows were proposed, potentially generating millions of safe and sustainable therapeutic products. Furthermore, clinical hECP doses were characterized as non-toxic in a standardized chorioallantoic membrane model. Lastly, a MACI-like protocol, including hECPs, was applied in a three-month GLP pilot safety evaluation in a caprine model of full-thickness articular cartilage defect. The safety of hECP transplantation was highlighted in xenogeneic settings, along with confirmed needs for optimal cell delivery vehicles and implantation techniques favoring effective cartilage repair or regeneration.

Combination of a Collagen Scaffold and an Adhesive Hyaluronan-Based Hydrogel for Cartilage Regeneration: A Proof of Concept in an Ovine Model.

OBJECTIVE: Hyaluronic acid-transglutaminase (HA-TG) is an enzymatically crosslinkable adhesive hydrogel with chondrogenic properties demonstrated in vitro and in an ectopic mouse model. In this study, we investigated the feasibility of using HA-TG in a collagen scaffold to treat chondral lesions in an ovine model, to evaluate cartilage regeneration in a mechanically and biologically challenging joint environment, and the influence of the surgical procedure on the repair process. DESIGN: Chondral defects of 6-mm diameter were created in the stifle joint of skeletally mature sheep. In a 3-month study, 6 defects were treated with HA-TG in a collagen scaffold to test the stability and biocompatibility of the defect filling. In a 6-month study, 6 sheep had 12 defects treated with HA-TG and collagen and 2 sheep had 4 untreated defects. Histologically observed quality of repair tissue and adjacent cartilage was semiquantitatively assessed. RESULTS: HA-TG adhered to the native tissue and did not cause any detectable negative reaction in the surrounding tissue. HA-TG in a collagen scaffold supported infiltration and chondrogenic differentiation of mesenchymal cells, which migrated from the subchondral bone through the calcified cartilage layer. Additionally, HA-TG and collagen treatment led to better adjacent cartilage preservation compared with empty defects (P < 0.05). CONCLUSIONS: This study demonstrates that the adhesive HA-TG hydrogel in a collagen scaffold shows good biocompatibility, supports in situ cartilage regeneration and preserves the surrounding cartilage. This proof-of-concept study shows the potential of this approach, which should be further considered in the treatment of cartilage lesions using a single-step procedure.

Virtual mechanical tests out-perform morphometric measures for assessment of mechanical stability of fracture healing in vivo.

Finite element analysis with models derived from computed tomography (CT) scans is potentially powerful as a translational research tool because it can achieve what animal studies and cadaver biomechanics cannot-low-risk, noninvasive, objective assessment of outcomes in living humans who have actually experienced the injury, or treatment being studied. The purpose of this study was to assess the validity of CT-based virtual mechanical testing with respect to physical biomechanical tests in a large animal model. Three different tibial osteotomy models were performed on 44 sheep. Data from 33 operated limbs and 20 intact limbs was retrospectively analyzed. Radiographic union scoring was performed on the operated limbs and physical torsional tests were performed on all limbs. Morphometric measures and finite element models were developed from CT scans and virtual torsional tests were performed to assess healing with four material assignment techniques. In correlation analysis, morphometric measures and radiographic scores were unreliable predictors of biomechanical rigidity, while the virtual torsion test results were strongly and significantly correlated with measured biomechanical test data, with high absolute agreement. Overall, the results validated the use of virtual mechanical testing as a reliable in vivo assessment of structural bone healing. This method is readily translatable to clinical evaluation for noninvasive assessment of the healing progress of fractures with minimal risk. Clinical significance: virtual mechanical testing can be used to reliably and noninvasively assess the rigidity of a healing fracture using clinical-resolution CT scans and that this measure is superior to morphometric and radiographic measures.

Imaging Modalities to Assess Fracture Healing.

PURPOSE OF REVIEW: This review discusses imaging modalities for fracture repair assessment, with an emphasis on pragmatic clinical and translational use, best practices for implementation, and challenges and opportunities for continuing research. RECENT FINDINGS: Semiquantitative radiographic union scoring remains the clinical gold standard, but has questionable reliability as a surrogate indicator of structural bone healing, particularly in early-stage, complex, or compromised healing scenarios. Alternatively, computed tomography (CT) scanning enables quantitative assessment of callus morphometry and mechanics through the use of patient-specific finite-element models. Dual-energy X-ray absorptiometry (DXA) scanning and radiostereometric analysis (RSA) are also quantitative, but technically challenging. Nonionizing magnetic resonance (MR) and ultrasound imaging are of high interest, but require development to enable quantification of 3D mineralized structures. Emerging image-based methods for quantitative assessment of bone healing may transform clinical research design by displacing binary outcomes classification (union/nonunion) and ultimately enhance clinical care by enabling early nonunion detection.