Chondrocyte-based intraoperative processing strategies for the biological augmentation of a polyurethane meniscus replacement.

Purpose/aim of study: Menisectomies account for over 1.5 million surgical interventions in Europe annually, and there is a growing interest in regenerative strategies to improve outcomes in meniscal replacement. The overall objective of this study was to evaluate the role of intraoperatively applied fresh chondrocyte (FC) isolates compared to minced cartilage (MC) fragments, used without cell isolation, to improve bioactivity and tissue integration when combined with a polyurethane replacement. MATERIALS AND METHODS: First, to optimize the intraoperative cell isolation protocol, caprine articular cartilage biopsies were digested with 750 U/ml or 3000 U/ml collagenase type II (ratio of 10 ml per g of tissue) for 30 min, 1 h or 12 h with constant agitation and compared to culture-expanded chondrocytes in terms of matrix deposition when cultured on polyurethane scaffolds. Finally, FCs and MC-augmented polyurethane scaffolds were evaluated in a caprine meniscal explant model to assess the potential enhancements on tissue integration strength. RESULTS: Adequate numbers of FCs were harvested using a 30 min chondrocyte isolation protocol and were found to demonstrate improved matrix deposition compared to standard culture-expanded cells in vitro. Upon evaluation in a meniscus explant defect model, both FCs and MC showed improved matrix deposition at the tissue-scaffold interface and enhanced push-out strength, fourfold and 2.5-fold, respectively, compared with the acellular implant. CONCLUSIONS: Herein, we have demonstrated a novel approach that could be applied intraoperatively, using FCs or MC for improved tissue integration with a polyurethane meniscal replacement.

Biomechanical properties of feline ventral abdominal wall and celiotomy closure techniques.

OBJECTIVE: To compare biomechanical properties and mechanism of failure of 3 regions of ventral abdominal wall in cats by using 2 suture materials, 2 suture bite-to-stitch intervals (SBSI), and full-thickness versus fascia-only closure. STUDY DESIGN: Randomized, cadaveric, ex vivo mechanical testing. SAMPLE POPULATION: 16 adult cat cadavers, 3 samples per cat. METHODS: Three regions of ventral abdominal wall were mechanically tested (N = 48 samples). Preumbilical, umbilical (U), and postumbilical (POU) regions were harvested by using a template. The thickness of the linea alba was recorded. Six samples without celiotomy served as controls. Twenty-eight samples were randomized to SBSI (2 × 2 or 5 × 5 mm) and suture material (3-0 polyglactin 910 or 3-0 polydioxanone) for simple continuous celiotomy closure. Fourteen samples were randomized to full-thickness or fascia-only closure. Samples were tested by linear distraction; tensile strength and mechanism of failure were recorded. Effects of body weight, thickness of linea alba, anatomic region, SBSI, type of closure, and suture material were evaluated by mixed model linear analysis. Load to failure was compared between males and females, full-thickness and fascia-only closure by independent t test, with P < .05 considered statistically significant. RESULTS: The POU region achieved lower loads to failure. Load to failure was greater in males compared with females. No difference was detected between full-thickness and fascia-only closure. Failure most commonly occurred by tearing of suture through tissues. Tissue failure with suture line loosening occurred mainly in the 5 × 5-mm SBSI group. CONCLUSION: The POU region is biomechanically weak and may therefore be predisposed to incisional herniation.

Extracellular matrix production by nucleus pulposus and bone marrow stem cells in response to altered oxygen and glucose microenvironments.

Bone marrow (BM) stem cells may be an ideal source of cells for intervertebral disc (IVD) regeneration. However, the harsh biochemical microenvironment of the IVD may significantly influence the biological and metabolic vitality of injected stem cells and impair their repair potential. This study investigated the viability and production of key matrix proteins by nucleus pulposus (NP) and BM stem cells cultured in the typical biochemical microenvironment of the IVD consisting of altered oxygen and glucose concentrations. Culture-expanded NP cells and BM stem cells were encapsulated in 1.5% alginate and ionically crosslinked to form cylindrical hydrogel constructs. Hydrogel constructs were maintained under different glucose concentrations (1, 5 and 25 mM) and external oxygen concentrations (5 and 20%). Cell viability was measured using the Live/Dead® assay and the production of sulphated glycosaminoglycans (sGAG), and collagen was quantified biochemically and histologically. For BM stem cells, IVD-like micro-environmental conditions (5 mM glucose and 5% oxygen) increased the accumulation of sGAG and collagen. In contrast, low glucose conditions (1 mM glucose) combined with 5% external oxygen concentration promoted cell death, inhibiting proliferation and the accumulation of sGAG and collagen. NP-encapsulated alginate constructs were relatively insensitive to oxygen concentration or glucose condition in that they accumulated similar amounts of sGAG under all conditions. Under IVD-like microenvironmental conditions, NP cells were found to have a lower glucose consumption rate compared with BM cells and may in fact be more suitable to adapt and sustain the harsh microenvironmental conditions. Considering the highly specialised microenvironment of the central NP, these results indicate that IVD-like concentrations of low glucose and low oxygen are critical and influential for the survival and biological behaviour of stem cells. Such findings may promote and accelerate the translational research of stem cells for the treatment of IVD degeneration.

Mechanical comparison of loop and crimp configurations for extracapsular stabilization of the cranial cruciate ligament-deficient stifle.

OBJECTIVE: To biomechanically compare different loop and crimp configurations used for extracapsular suture stabilization of the CCL-deficient stifle. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Crimped loop constructs of 100 lb Ande type nylon leader line in 7 different configurations comprising single and double loops, single and double crimps, and the interlocking loop configuration. METHODS: Constructs premade on external skeletal fixator bars 60 mm apart and tested in tension with a custom-made split circular arm mounted on a table-top materials testing machine. Data were derived from force/displacement plots. In "load to failure" test (10/group) constructs were loaded to failure with distraction rates of 10 mm/min; ultimate load, tension at 2 mm elongation and failure were recorded. In "staircase" test (5/group) constructs cycled at 100 N/s from 75 N with incremental increases of 50 N/cycle; ultimate load, maximum tension before elongation at rest over 2 mm and failure were recorded. In "cycling and jumping" test (10/group) 3 of 7 constructs cycled at 100 N/s 100 times from 50 to 100 N, then 5 times from 50 to 600 N; failure and elongation at cycles 1, 50 and 100 and at jumps 1 to 5 recorded. RESULTS: Double-loop double-crimp configurations were statistically superior to all other configurations in ultimate load, and to single-loop and interlocking loop configurations in elongation in "load to failure" and "staircase" tests. In "cycling and jumping" test the interlocking loop configuration specimens elongated significantly more than the others and only in the double-loop double-crimp group did all constructs complete the test. CONCLUSIONS: Double-loop double-crimp configurations are mechanically superior to other previously described configurations.

Knot Security of 5 Metric (USP 2) Sutures: Influence of Knotting Technique, Suture Material, and Incubation Time for 14 and 28 Days in Phosphate Buffered Saline and Inflamed Equine Peritoneal Fluid.

OBJECTIVES: To evaluate knot security for 3 knot types created in 3 commonly used 5 metric suture materials incubated in physiological and pathological fluids. STUDY DESIGN: In vitro mechanical study. SAMPLE POPULATION: Knotted suture loops (n = 5/group). METHODS: Loops of 3 different suture materials (glycolide/lactide copolymer; polyglactin 910; polydioxanone) were created around a 20 mm rod using 3 knot types (square [SQ], surgeon's [SK], and triple knot [TK]) and were tested to failure in distraction (6 mm/min) after tying (day 0) and after being incubated for 14 and 28 days in phosphate buffered saline (PBS) or inflamed peritoneal fluid. Failure load (N) and mode were recorded and compared. RESULTS: For polydioxanone, significant differences in force to knot failure were found between SQ and SK/TK but not between SK and TK. The force required to break all constructs increased after incubation in phosphate buffered saline (PBS). With glycolide/lactide copolymer no differences in force to knot failure were observed. With polyglactin 910, a significant difference between SQ and TK was observed, which was not seen between the other knot types. Incubation in inflamed peritoneal fluid caused a larger and more rapid decrease in force required to cause knot failure than incubation in PBS. CONCLUSIONS: Mechanical properties of suture materials have significant effects on knot security. For polydioxanone, SQ is insufficient to create a secure knot. Additional wraps above a SK confer extra stability in some materials, but this increase may not be clinically relevant or justifiable. Glycolide/lactide copolymer had excellent knot security.

Engineering articular cartilage-like grafts by self-assembly of infrapatellar fat pad-derived stem cells.

Well documented limitations associated with primary chondrocytes for cartilage tissue engineering applications have led to increased interest in the use of multi-potent stem/progenitor cells. The objective of this study was to firstly investigate if infrapatellar fat pad-derived stem cells (FPSCs) could be used to engineer cartilage-like tissues through a self-assembly (SA) process, and secondly to compare the properties of such grafts to those engineered by agarose hydrogel encapsulation (AE). Self-assembled cartilaginous tissues were first engineered by geometrically confining FPSCs on tissue culture plastic, and then either continuously or transiently supplementing these constructs with transforming growth factor-b3 (TGF-b3). Transient supplementation with TGF-b3 (for the first 21 days of culture) enhanced the development of self-assembled grafts, with sGAG accumulation reaching levels of 8.4 ± 1.5% w/w after 6 weeks of culture. While overall levels of matrix synthesis were higher with AE compared to SA, when normalized to tissue wet weight, ECM accumulation was significantly greater in the lighter SA constructs. A potential drawback with the SA approach on tissue culture plastic was that it often led to the development of contracted,geometrically inconsistent tissues.We therefore next explored if SA on polyethylene terephthalate (PET) transwell membranes would lead to the development of more morphologically stable and homogenous tissues. At high seeding densities, SA on such transwell membranes led to the formation of geometrically uniform constructs that underwent minimal contraction during culture. In conclusion, the results of this study demonstrate the potential of SA using FPSCs for cartilage tissue engineering, with grafts attaining relatively high levels of sGAG content within clinically relevant timeframes. Such an approach is easily scalable and may lend itself to treating large, full thickness cartilage defects.

Development and characterization of antacid microcapsules to buffer the acidic intervertebral disc microenvironment.

During intervertebral disc (IVD) degeneration, microenvironmental challenges such as decreasing levels of glucose, oxygen, and pH play crucial roles in cell survival and matrix turnover. Antacids, such as Mg(OH)2 and CaCO3, entrapped in microcapsules are capable of neutralizing acidic microenvironments in a controlled fashion and therefore may offer the potential to improve the acidic niche of the degenerated IVD and enhance cell-based regeneration strategies. The objectives of this work were, first, to develop and characterize antacid microcapsules and assess their neutralization capacity in an acidic microenvironment and, second, to combine antacid microcapsules with cellular microcapsules in a hybrid gel system to investigate their neutralization effect as a potential therapeutic in a disc explant model. To achieve this, we screened five different pH- neutralizing agents (Al(OH)3, Mg(OH)2, CaCO3, and HEPES) in terms of their pH neutralization capacities, with Mg(OH)2 or CaCO3 being carried forward for further investigation. Antacid-alginate microcapsules were formed at different concentrations using the electrohydrodynamic spraying process and assessed in terms of size, buffering kinetics, cell compatibility, and cytotoxicity. Finally, the combination of cellular microcapsules and antacid capsules was examined in a bovine disc explant model under physiological degenerative conditions. Overall, CaCO3 was found to be superior in terms of neutralization capacities, release kinetics, and cellular response. Specifically, CaCO3 elevated the acidic pH to neutral levels and is estimated to be maintained for several weeks based on Ca2+ release. Using a disc explant model, it was demonstrated that CaCO3 microcapsules were capable of increasing the local pH within the core of a hybrid cellular gel system. This work highlights the potential of antacid microcapsules to positively alter the challenging acidic microenvironment conditions typically observed in degenerative disc disease, which may be used in conjunction with cell therapies to augment regeneration.

The influence of pH and salt concentration on the microstructure and mechanical properties of meniscus extracellular matrix-derived implants.

Meniscus-related injuries are a common orthopedic challenge with an increasing incidence in the population. While the preservation of viable meniscal tissue is the preferred approach in repair strategies, complex or total traumatic lesions may require alternative therapeutic approaches such as meniscal reconstruction using allografts or engineered equivalents. Although clinical studies suggest promising outcomes with the use of acellular implants, further development is needed to improve their biological and mechanical requirements. Decellularized extracellular matrix (dECM) derived from menisci is a promising biomaterial for meniscus tissue engineering due to its recapitulation of the native tissue environment and the maintenance of tissue-specific cues. However, the associated mechanical limitations of dECM-derived scaffolds frequently impedes their adoption, requiring additional reinforcement or combining with stiffer biomaterials to increase their load-bearing properties. In this study, decellularized extracellular matrix was extracted and its fibrillation was controlled by adjusting both pH and salt concentrations to fabricate mechanically functional meniscal tissue equivalents. The effect of collagen fibrillation on the mechanical properties of the dECM constructs was assessed, and porcine-derived fibrochondrocytes were used to evaluate in vitro biocompatibility. It was also possible to fabricate meniscus-shaped implants by casting of the dECM and to render the implants suitable for off-the-shelf use by adopting a freeze-drying preservation method. Suture pull-out tests were also performed to assess the feasibility of using existing surgical methods to fix such implants within a damaged meniscus. This study highlights the potential of utilizing ECM-derived materials for meniscal tissue substitutes that closely mimic the mechanical and biological properties of native tissue.

Preclinical to clinical translation for intervertebral disc repair: Effects of species-specific scale, metabolism, and matrix synthesis rates on cell-based regeneration.

Background: A significant hurdle for potential cell-based therapies is the subsequent survival and regenerative capacity of implanted cells. While many exciting developments have demonstrated promise preclinically, cell-based therapies for intervertebral disc (IVD) degeneration fail to translate equivalent clinical efficacy. Aims: This work aims to ascertain the clinical relevance of both a small and large animal model by experimentally investigating and comparing these animal models to human from the perspective of anatomical scale and their cellular metabolic and regenerative potential. Materials and Methods: First, this work experimentally investigated species-specific geometrical scale, native cell density, nutrient metabolism, and matrix synthesis rates for rat, goat, and human disc cells in a 3D microspheroid configuration. Second, these parameters were employed in silico to elucidate species-specific nutrient microenvironments and predict differences in temporal regeneration between animal models. Results: This work presents in silico models which correlate favorably to preclinical literature in terms of the capabilities of animal regeneration and predict that compromised nutrition is not a significant challenge in small animal discs. On the contrary, it highlights a very fine clinical balance between an adequate cell dose for sufficient repair, through de novo matrix deposition, without exacerbating the human microenvironmental niche. Discussion: Overall, this work aims to provide a path towards understanding the effect of cell injection number on the nutrient microenvironment and the "time to regeneration" between preclinical animal models and the large human IVD. While these findings help to explain failed translation of promising preclinical data and the limited results emerging from clinical trials at present, they also enable the research field and clinicians to manage expectations on cell-based regeneration. Conclusion: Ultimately, this work provides a platform to inform the design of clinical trials, and as computing power and software capabilities increase in the future, it is conceivable that generation of patient-specific models could be used for patient assessment, as well as pre- and intraoperative planning.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells.

The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO(2)) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO(2)). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO(2) proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO(2) also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO(2) was found to be a more potent promoter of chondrogenesis than expansion at 5% pO(2). Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO(2) compared to 5% pO(2). Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO(2) also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a chondrogenic phenotype for use in cartilage repair therapies or to promote hypertrophy of cartilaginous grafts for endochondral bone repair strategies.

Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells.

Hydrostatic pressure (HP) is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-ß3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-ß3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-ß3 stimulation. HP in the absence of TGF-ß3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-ß3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-ß3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

Consolidating and re-evaluating the human disc nutrient microenvironment.

Background: Despite exciting advances in regenerative medicine, cell-based strategies for treating degenerative disc disease remain in their infancy. To maximize the potential for successful clinical translation, a more thorough understanding of the in vivo microenvironment is needed to better determine and predict how cell therapies will respond when administered in vivo. Aims: This work aims to reflect on the in vivo nutrient microenvironment of the degenerating IVD through consolidating what has already been measured together with investigative in silico models. Materials and Methods: This work uses in silico modeling, underpinned by more recent experimentally determined parameters of degeneration and nutrient transport from the literature, to re-evaluate the current knowledge in terms of grade-specific stages of degeneration. Results: Through modeling only the metabolically active cell population, this work predicts slightly higher glucose concentrations compared to previous in silico models, while the predicted results show good agreement with previous intradiscal pH and oxygen measurements. Increasing calcification with degeneration limits nutrient transport into the IVD and initiates a build-up of acidity; however, its effect is compensated somewhat by a reduction in diffusional distance due to decreasing disc height. Discussion: This work advances in silico modeling through a strong foundation of experimentally determined grade-specific input parameters. Taken together, pre-existing measurements and predicted results suggest that metabolite concentrations may not be as critically low as commonly believed, with calcification not appearing to have a detrimental effect at stages of degeneration when cell therapies are an appropriate intervention. Conclusion: Overall, our initiative is to provoke greater deliberation and consideration of the nutrient microenvironment when performing in vitro cell culture and cell therapy development. This work highlights urgency for robust experimental glucose measurements in healthy and degenerating IVDs, not only to validate in silico models but to significantly advance the field in fully elucidating the nutrient microenvironment and refining in vitro techniques to accelerate clinical translation.

Two- and three-dimensional in vitro nucleus pulposus cultures: An in silico analysis of local nutrient microenvironments.

Background: It is well established that the unique biochemical microenvironment of the intervertebral disc plays a predominant role in cell viability and biosynthesis. However, unless the effect of microenvironmental conditions is primary to a study objective, in vitro culture parameters that are critical for reproducibility are both varied and not routinely reported. Aims: This work aims to investigate the local microenvironments of commonly used culture configurations, highlighting physiological relevance, potential discrepancies, and elucidating possible heterogeneity across the research field. Materials and Methods: This work uses nutrient-transport in silico models to reflect on the effect of often underappreciated parameters, such as culture geometry and diffusional distance (vessel, media volume, construct size), seeding density, and external boundary conditions on the local microenvironment of two-dimensional (2D) and three-dimensional (3D) in vitro culture systems. Results: We elucidate important discrepancies between the external boundary conditions such as the incubator level or media concentrations and the actual local cellular concentrations. Oxygen concentration and cell seeding density were found to be highly influential parameters and require utmost consideration when utilizing 3D culture systems. Discussion: This work highlights that large variations in the local nutrient microenvironment can easily be established without consideration of several key parameters. Without careful deliberation of the microenvironment within each specific and unique system, there is the potential to confound in vitro results leading to heterogeneous results across the research field in terms of biosynthesis and matrix composition. Conclusion: Overall, this calls for a greater appreciation of key parameters when designing in vitro experiments. Better harmony and standardization of physiologically relevant local microenvironments are needed to push toward reproducibility and successful translation of findings across the research field.

Rat tail models for the assessment of injectable nucleus pulposus regeneration strategies.

Back pain is a global epidemiological and socioeconomic problem often associated with intervertebral disc degeneration; a condition believed to initiate in the nucleus pulposus (NP). There is considerable interest in developing early therapeutic interventions to target the NP and halt degeneration. Rat caudal models of disc degeneration have demonstrated significant utility in the study of disease progression and its impact on tissue structure, composition, and mechanical performance. One significant advantage of the caudal model is the ease of access and high throughput nature. However, considerable variability exists across the literature in terms of experimental setup and parameters. The objective of this article is to aid researchers in the design and development of caudal puncture models by providing details and insight into the most reported experimental parameters. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were employed to screen the existing literature and 80 manuscripts met the inclusion criteria. Disc geometry, surgical approaches, effect of needle gauge size to induce degeneration, therapeutic volume, outcome measures, and associated limitations are considered and discussed, and a range of recommendations based on different research questions are presented.

Promoting endogenous articular cartilage regeneration using extracellular matrix scaffolds.

Articular cartilage defects fail to heal spontaneously, typically progressing to osteoarthritis. Bone marrow stimulation techniques such as microfracture (MFX) are the current surgical standard of care; however MFX typically produces an inferior fibro-cartilaginous tissue which provides only temporary symptomatic relief. Here we implanted solubilised articular cartilage extracellular matrix (ECM) derived scaffolds into critically sized chondral defects in goats, securely anchoring these implants to the joint surface using a 3D-printed fixation device that overcame the need for sutures or glues. In vitro these ECM scaffolds were found to be inherently chondro-inductive, while in vivo they promoted superior articular cartilage regeneration compared to microfracture. In an attempt to further improve the quality of repair, we loaded these scaffolds with a known chemotactic factor, transforming growth factor (TGF)-ß3. In vivo such TGF-ß3 loaded scaffolds promoted superior articular cartilage regeneration. This study demonstrates that ECM derived biomaterials, either alone and particularly when combined with exogenous growth factors, can successfully treat articular cartilage defects in a clinically relevant large animal model.

Multi-factorial nerve guidance conduit engineering improves outcomes in inflammation, angiogenesis and large defect nerve repair.

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.

Bilayered extracellular matrix derived scaffolds with anisotropic pore architecture guide tissue organization during osteochondral defect repair.

While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.

A growth factor delivery system for chondrogenic induction of infrapatellar fat pad-derived stem cells in fibrin hydrogels.

Articular cartilage has a limited capacity for self-renewal and repair. Tissue engineering of cartilage in vitro has been proposed as a solution to this problem; however, this approach is costly and requires a significant amount of time to grow the graft. An alternative approach is to implant chondroprogenitor cells seeded within a growth factor delivery scaffold directly into the defect site to promote tissue regeneration. The objective of this study was to develop a biocompatible growth factor delivery system capable of promoting chondrogenesis of infrapatellar fat pad (IFP)-derived stem cells. Transforming growth factor beta-1 (TGF-ß1) was loaded into gelatin microspheres and incorporated into fibrin hydrogels containing IFP-derived stem cells. The release of TGF-ß1 was quantified using an enzyme-linked immunosorbent assay, whereas chondrogenesis was demonstrated histologically and by quantifying sulfated glycosaminoglycan production after 21 days of in vitro culture. TGF-ß1 loaded into gelatin microspheres appeared to be as effective in promoting chondrogenesis of IFP-derived stem cells as adding TGF-ß1 directly to the medium. The influence of different microsphere fabrication parameters and TGF-ß1 loading concentrations was also investigated but appeared to only have a small effect on subsequent chondrogenesis. The development of such growth factor delivery systems in combination with IFP-derived stem cells represents a potential new strategy for cartilage defect repair.

Investigating the physiological relevance of ex vivo disc organ culture nutrient microenvironments using in silico modeling and experimental validation.

BACKGROUND: Ex vivo disc organ culture systems have become a valuable tool for the development and pre-clinical testing of potential intervertebral disc (IVD) regeneration strategies. Bovine caudal discs have been widely selected due to their large availability and comparability to human IVDs in terms of size and biochemical composition. However, despite their extensive use, it remains to be elucidated whether their nutrient microenvironment is comparable to human degeneration. AIMS: This work aims to create the first experimentally validated in silico model which can be used to predict and characterize the metabolite concentrations within ex vivo culture systems. MATERIALS & METHODS: Finite element models of cultured discs governed by previously established coupled reaction-diffusion equations were created using COMSOL Multiphysics. Experimental validation was performed by measuring oxygen, glucose and pH levels within discs cultured for 7 days, in a static compression bioreactor. RESULTS: The in silico model was successfully validated through good agreement between the predicted and experimentally measured concentrations. For an ex vivo organ cultured in high glucose medium (4.5 g/L or 25 mM) and normoxia, a larger bovine caudal disc (Cd1-2 to Cd3-4) had a central concentration of ~2.6 %O2, ~8 mM of glucose and a pH value of 6.7, while the smallest caudal discs investigated (Cd6-7 and Cd7-8), had a central concentration of ~6.5 %O2, ~12 mM of glucose and a pH value of 6.9. DISCUSSION: This work advances the knowledge of ex vivo disc culture microenvironments and highlights a critical need for optimization and standardization of culturing conditions. CONCLUSION: Ultimately, for assessment of cell-based therapies and successful clinical translation based on nutritional demands, it is imperative that the critical metabolite values within organ cultures (minimum glucose, oxygen and pH values) are physiologically relevant and comparable to the stages of human degeneration.