a-rat-tibial-growth-plate-injury-model-to-characterize-repair

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

fabrication-size-controlled-emulsion-free-chitosan-genipin-microgels

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Gates Center for Regenerative Medicine, University of Colorado Anschutz Medical Campus

University of Colorado Anschutz Medical Campus

In this work, an emulsion crosslinking method was developed to produce chitosan-genipin microgels which acted as an injectable and microporous scaffold. Chitosan was characterized with respect to pH by light scattering and aqueous titration. Microgels were characterized with swelling, light scattering, and rheometry of densely-packed microgel solutions. The results suggest that as chitosan becomes increasingly deprotonated above the pKa, repulsive forces diminish and intermolecular attractions cause pH-responsive chain aggregation; leading to microgel-microgel aggregation as well. The microgels with the most chitosan and least cross-linker showed the highest yield stress and a storage modulus of 16kPa when condensed as a microgel paste at pH 7.4. Two oppositely-charged growth factors could be encapsulated into the microgels and endothelial cells were able to proliferate into the 3D microgel scaffold. This work motivates further research on the applications of the chitosan microgel scaffold as an injectable and microporous scaffold in regenerative medicine.

Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.

The physis, or growth plate, is a cartilaginous region at the end of children's long bones that serves as the primary center for longitudinal growth and characterizes the immature skeleton. Musculoskeletal injury, including fracture, infection, malignancy, or iatrogenic damage, has risk of physeal damage. Physeal injuries account for 30% of pediatric fractures and may result in impaired bone growth. Once damaged, cartilage tissue within the physis is often replaced by unwanted bony tissue, forming a "bony bar" that can lead to complications such as complete growth arrest, angular or rotational deformities, and altered joint mechanics. Children with a bony bar occupying <50% of the physis usually undergo bony bar resection and insertion of an interpositional material, such as a fat graft, to prevent recurrence and allow the surrounding uninjured physeal tissue to restore longitudinal bone growth. Clinical success for this procedure is <35% and often the bony bar and associated growth impairments return. Children who are not candidates for bony bar resection due to a physeal bar occupying >50% of their physis undergo corrective osteotomy or bone lengthening procedures. These approaches are complex and have variable success rates. As such, there is a critical need for regenerative approaches to not only prevent initial bony bar formation but also regenerate healthy physeal cartilage following injury. This review describes physeal anatomy, mechanisms of physeal injury, and current treatment options with associated limitations. Furthermore, we provide an overview of the current research using cell-based therapies, growth factors, and biomaterials in the different animal models of injury along with strategic directions for modulating intrinsic injury pathways to inhibit bony bar formation and/or promote physeal tissue formation. Pediatric physeal injuries constitute a unique niche within regenerative medicine for which there is a critical need for research to decrease child morbidity related to this injurious process.

Regenerative Medicine Approaches for the Treatment of Pediatric Physeal Injuries.

Chondrolabral damage is commonly observed in patients with cam-type femoroacetabular impingement (FAI). Chondral flap reattachment has recently been proposed as a possible preservation technique.

Viability and Tissue Quality of Cartilage Flaps From Patients With Femoroacetabular Hip Impingement: A Matched-Control Comparison.

Focal chondral lesions and early osteoarthritis (OA) are responsible for progressive joint pain and disability in millions of people worldwide, yet there is currently no surgical joint preservation treatment available to fully restore the long term functionality of cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. Toward improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Injectable hydrogels have emerged as a promising scaffold due to their wide range of properties, the ability to encapsulate cells within the material, and their ability to provide cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi-layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarises the innovative approaches to engineer injectable hydrogels toward cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:64-75, 2018.

Current and novel injectable hydrogels to treat focal chondral lesions: Properties and applicability.

In this study, we investigate the in vitro and in vivo chondrogenic capacity of a novel photopolymerizable cartilage mimetic hydrogel, enhanced with extracellular matrix analogs, for cartilage regeneration.

Photopolymerizable Injectable Cartilage Mimetic Hydrogel for the Treatment of Focal Chondral Lesions: A Proof of Concept Study in a Rabbit Animal Model.

Biopolymer microgels are emerging as a versatile tool for aiding in the regeneration of damaged tissues due to their biocompatible nature, tunable microporous structure, ability to encapsulate bioactive factors, and tailorable properties such as stiffness and composition. These properties of microgels, along with their injectability, have allowed for their utilization in a multitude of different tissue engineering applications. Controlled release of growth factors, antibodies, and other bioactive factors from microgels have demonstrated their capabilities as transporters for essential bioactive molecules necessary for guiding tissue reconstruction. Additionally, recent in vitro studies of cellular interaction and proliferation within microgel structures have laid the initial groundwork for regenerative tissue engineering using these materials. Microgels have even been crosslinked together in various ways or 3D printed to form three-dimensional scaffolds to support cell growth. In vivo studies of microgels have pioneered the clinical relevance of these novel and innovative materials for regenerative tissue engineering. This review will cover recent developments and research of microgels as they pertain to bioactive factor release, cellular interaction and proliferation in vitro, and tissue regeneration in vivo. STATEMENT OF SIGNIFICANCE: This review is focused on state-of-the-art microgel technology and innovations within the tissue engineering field, focusing on the use of microgels in bioactive factor delivery and as cell-interactive scaffolds, both in vitro and in vivo. Microgels are hydrogel microparticles that can be tuned based on the biopolymer from which they are derived, the crosslinking chemistry used, and the fabrication method. The emergence of microgels for tissue regeneration applications in recent years illuminates their versatility and applicability in clinical settings.

Microgels: Modular, tunable constructs for tissue regeneration.

Physeal injuries can lead to bony repair tissue formation, known as a bony bar. This can result in growth arrest or angular deformity, which is devastating for children who have not yet reached their full height. Current clinical treatment involves resecting the bony bar and replacing it with a fat graft to prevent further bone formation and growth disturbance, but these treatments frequently fail to do so and require additional interventions. Novel treatments that could prevent bone formation but also regenerate the injured physeal cartilage and restore normal bone elongation are warranted. To test the efficacy of these treatments, animal models that emulate human physeal injury are necessary. The rabbit model of physeal injury quickly establishes a bony bar, which can then be resected to test new treatments. Although numerous rabbit models have been reported, they vary in terms of size and location of the injury, tools used to create the injury, and methods to assess the repair tissue, making comparisons between studies difficult. The study presented here provides a detailed method to create a rabbit model of proximal tibia physeal injury using a two-stage procedure. The first procedure involves unilateral removal of 25% of the physis in a 6-week-old New Zealand white rabbit. This consistently leads to a bony bar, significant limb length discrepancy, and angular deformity within 3 weeks. The second surgical procedure involves bony bar resection and treatment. In this study, we tested the implantation of a fat graft and a photopolymerizable hydrogel as a proof of concept that injectable materials could be delivered into this type of injury. At 8 weeks post-treatment, we measured limb length, tibial angle, and performed imaging and histology of the repair tissue. By providing a detailed, easy to reproduce methodology to perform the physeal injury and test novel treatments after bony bar resection, comparisons between studies can be made and facilitate translation of promising therapies toward clinical use. Impact Statement This study provides details to create a rabbit model of physeal injury that can facilitate comparisons between studies and test novel regenerative medicine approaches. Furthermore, this model mimics the human, clinical situation that requires a bony bar resection followed by treatment. In addition, identification of a suitable treatment can be seen in the correction of the growth deformity, allowing this model to facilitate the development of novel physeal cartilage regenerative medicine approaches.

Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches.

The physis is a cartilaginous tissue in children's long bones that is responsible for bone elongation. Physeal injuries can heal with bony repair tissue known as a "bony bar," and this can cause growth deformities. Current treatments involve surgical resection of the bony bar and insertion of inert materials in hopes of preventing bony bar re-formation and preserving bone elongation. However, these materials frequently fail and the bony bar commonly returns. This study investigated alginate-chitosan hydrogels as interpositional materials to block bony bar formation in a rat model of physeal injury. Further, biomaterial properties such as substrate stiffness, permeability, and degradation rate were studied. Different ratio alginate:chitosan hydrogels with or without calcium cross-linking were tested for their inhibition of bony bar formation and restoration of the injured physis. Alginate:chitosan were mixed (a) 90:10 with calcium (90:10 + Ca); (b) 50:50 with calcium (50:50 + Ca); (c) 50:50 without calcium (50:50 - Ca); and (d) 50:50 made with irradiated alginate (IA) and without calcium. We found that repair tissue was determined primarily by the in vivo degradation rate of alginate-chitosan hydrogels. 90:10 + Ca had a slow degradation rate, prevented cellular infiltration, and produced the most bony bar tissue while having softer, more permeable material properties. IA had the fastest degradation, showed high cellular infiltration, and produced the most cartilage-like tissue while having stiffer, less permeable material properties. Our results suggest that the in vivo biomaterial degradation rate is a dynamic property that can be optimized to influence cell fate and tissue repair in physeal injuries.

In vivo degradation rate of alginate-chitosan hydrogels influences tissue repair following physeal injury.

Physeal injuries can result in the formation of a "bony bar" which can lead to bone growth arrest and deformities in children. Vascular endothelial growth factor (VEGF) has been shown to play a role in bony bar formation, making it a potential target to inhibit bony repair tissue after physeal injury. The goal of this study was to investigate whether the local delivery of anti-VEGF antibody (α-VEGF; 7.5 μg) from alginate:chitosan hydrogels to the tibial physeal injury site in rats prevents bony bar formation. We tested the effects of quick or delayed delivery of α-VEGF using both 90:10 and 50:50 ratio alginate:chitosan hydrogels, respectively. Male and female 6-week-old Sprague-Dawley rats received a tibial physeal injury and the injured site injected with alginate-chitosan hydrogels: (1) 90:10 (Quick Release); (2) 90:10 + α-VEGF (Quick Release + α-VEGF); (3) 50:50 (Slow Release); (4) 50:50 +  α-VEGF (Slow Release +  α-VEGF); or (5) Untreated. At 2, 4, and 24 weeks postinjury, animals were euthanized and tibiae assessed for bony bar and vessel formation, repair tissue type, and limb lengthening. Our results indicate that Quick Release + α-VEGF reduced bony bar and vessel formation, while also increasing cartilage repair tissue. Further, the quick release of α-VEGF neither affected limb lengthening nor caused deleterious side-effects in the adjacent, uninjured physis. This α-VEGF treatment, which inhibits bony bar formation without interfering with normal bone elongation, could have positive implications for children suffering from physeal injuries.

Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats.

The growth plate is a cartilage tissue near the ends of children's long bones and is responsible for bone growth. Injury to the growth plate can result in the formation of a 'bony bar' which can span the growth plate and result in bone growth abnormalities in children. Biomaterials such as chitosan microgels could be a potential treatment for growth plate injuries due to their chondrogenic properties, which can be enhanced through loading with biologics. They are commonly fabricated via an emulsion method, which involves solvent rinses that are cytotoxic. Here, we present a high throughput, non-cytotoxic, non-emulsion-based method to fabricate chitosan-genipin microgels. Chitosan was crosslinked with genipin to form a hydrogel network, and then pressed through a syringe filter using mesh with various pore sizes to produce a range of microgel particle sizes. The microgels were then loaded with chemokines and growth factors and their release was studied in vitro. To assess the applicability of the microgels for growth plate cartilage regeneration, they were injected into a rat growth plate injury. They led to increased cartilage repair tissue and were fully degraded by 28 days in vivo. This work demonstrates that chitosan microgels can be fabricated without solvent rinses and demonstrates their potential for the treatment of growth plate injuries.

Emulsion-free chitosan-genipin microgels for growth plate cartilage regeneration.

RNA-sequencing (RNA-seq) is a novel and highly sought-after tool in the field of musculoskeletal regenerative medicine. The technology is being used to better understand pathological processes, as well as elucidate mechanisms governing development and regeneration. It has allowed in-depth characterization of stem cell populations and discovery of molecular mechanisms that regulate stem cell development, maintenance, and differentiation in a way that was not possible with previous technology. This review introduces RNA-seq technology and how it has paved the way for advances in musculoskeletal regenerative medicine.

Understanding the Transcriptomic Landscape to Drive New Innovations in Musculoskeletal Regenerative Medicine.

The growth plate is a cartilaginous tissue that functions to lengthen bones in children. When fractured, however, the growth plate can lose this critical function. Our understanding of growth plate fracture and mechanobiology is currently hindered by sparse information on the growth plate's microscale spatial gradients in mechanical properties. In this study, we performed microindentation across the proximal tibia growth plate of 9-week-old New Zealand White rabbits (n = 15) to characterize spatial variations in mechanical properties using linear elastic and nonlinear poroelastic material models. Mean indentation results for Hertz reduced modulus ranged from 380 to 690 kPa, with a peak in the upper hypertrophic zone and significant differences (p < 0.05) between neighboring zones. Using a subset of these animals (n = 7), we characterized zonal structure and extracellular matrix content of the growth plate through confocal fluorescent microscopy and Raman spectroscopy mapping. Comparison between mechanical properties and matrix content across the growth plate showed that proteoglycan content correlated with compressive modulus. This study is the first to measure poroelastic mechanical properties from microindentation across growth plate cartilage and to discern differing mechanical properties between the upper and lower hypertrophic zones. This latter finding may explain the location of typical growth plate fractures. The spatial variation in our reported mechanical properties emphasize the heterogeneous structure of the growth plate which is important to inform future regenerative implant design and mechanobiological models.

The heterogeneous mechanical properties of adolescent growth plate cartilage: A study in rabbit.

The aim of this study was to assess the bulk material properties and depth-dependent strain distribution of bovine growth plate cartilage. We hypothesized that both moduli and strain distribution are highly depth-, orientation-, and location-dependent. Bovine proximal tibiae (1-month-old) were sliced along the sagittal and coronal planes to create ∼ 4 mm samples. Digital image correlation (DIC) was combined with stress relaxation tests for evaluation of bulk modulus (tangent and equilibrium) and depth-dependent strain distribution. A subset of samples was imaged after Col-F staining as well as histological staining (Safranin-O/Fast Green) to evaluate zonal organization and matrix composition. The mean tangent modulus was 4.25 ± 2.46 MPa while the equilibrium modulus was 0.86 ± 0.46 MPa. No significant differences in moduli were found with respect to orientation (sagittal vs coronal face), but sagittal location within the joint was a significant predictor for tangent modulus. Overall moduli values decreased from the periphery to the midline of the joint. Depth-dependent cellular organization, determined by cell density and shape, was highly variable. This heterogeneity may be a biological toughening mechanism. Peak normalized strains were observed most often in the hypertrophic zone. Modulus was significantly lower in the hypertrophic zone as compared to the resting and proliferative zones. This study is the first to evaluate moduli and strain distribution in intact growth plates as a function of depth, orientation, and anatomic location. Future work with growth plate tissue engineering should consider the location- and depth-dependent nature of the native tissue mechanical properties when designing mimetic constructs.

ArticleTotal : 2	Year
Publication title Cited by 21	2017
Publication title Cited by 9	2022

Article	Year
Injectable and microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels. Carbohydrate polymers\| PubMed ID: 27516331	2016
Regenerative Medicine Approaches for the Treatment of Pediatric Physeal Injuries. Tissue engineering. Part B, Reviews\| PubMed ID: 28830302	2018
Viability and Tissue Quality of Cartilage Flaps From Patients With Femoroacetabular Hip Impingement: A Matched-Control Comparison. Orthopaedic journal of sports medicine\| PubMed ID: 28868322	2017
Current and novel injectable hydrogels to treat focal chondral lesions: Properties and applicability. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 28975658	2018
Photopolymerizable Injectable Cartilage Mimetic Hydrogel for the Treatment of Focal Chondral Lesions: A Proof of Concept Study in a Rabbit Animal Model. The American journal of sports medicine\| PubMed ID: 30481048	2019
Microgels: Modular, tunable constructs for tissue regeneration. Acta biomaterialia\| PubMed ID: 30769137	2019
Rabbit Model of Physeal Injury for the Evaluation of Regenerative Medicine Approaches. Tissue engineering. Part C, Methods\| PubMed ID: 31552802	2019
In vivo degradation rate of alginate-chitosan hydrogels influences tissue repair following physeal injury. Journal of biomedical materials research. Part B, Applied biomaterials\| PubMed ID: 32034881	2020
Anti-VEGF antibody delivered locally reduces bony bar formation following physeal injury in rats. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 33179297	2021
Emulsion-free chitosan-genipin microgels for growth plate cartilage regeneration. Journal of biomaterials applications\| PubMed ID: 33709832	2021
Understanding the Transcriptomic Landscape to Drive New Innovations in Musculoskeletal Regenerative Medicine. Current osteoporosis reports\| PubMed ID: 35156183	2022
The heterogeneous mechanical properties of adolescent growth plate cartilage: A study in rabbit. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 35203020	2022
Material properties and strain distribution patterns of bovine growth plate cartilage vary with anatomic location and depth. Journal of biomechanics\| PubMed ID: 35245713	2022

Karin A. Payne

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