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The natural bone repair processes are sufficient to effect timely restoration of skeletal integrity for most fractures when an appropriate mechanical environment exists or is created with internal fixation or coaptation. However, some situations require manipulation or augmentation of natural healing mechanisms to regenerate larger quantities of new bone than would naturally occur to achieve surgical goals. Examples include substantial loss of host bone from trauma, arthrodesis, non- or delayed unions, or insufficient healing potential of the host because of local or systemic disease, e.g. bone infection or osteocyst like lesions.Materials and strategies that are employed must duplicate and amplify the events of secondary bony formation to achieve the desired result.Bone can be regenerated through the following strategies: osteogenesis—the transfer of cells; osteoinduction—the induction of cells to become bone; osteoconduction, providing a scaffold for bone forming cells; or osteopromotion—the promotion of bone healing and regeneration by encouraging the biologic or mechanical environment of the healing or regenerating tissues. The most efficacious strategies use as many of these fundamental components of bone regeneration as possible (Figure 1). Table 1: Definition of functions of bone grafts and synthetic bone replacements. Bone graftsType: The gold standard for augmenting bone healing in humans and other animals remains autogenous cancellous bone graft. More than 500,000 bone grafting procedures are performed annually in human patients in the United States, and 2.2 million are completed worldwide. The number performed in companion animals, while undocumented, is also likely to be substantial.Classification: Bone grafts are typically classified according to the origin (i.e., autograft, allograft or xenograft), tissue type (i.e. cancellous, cortical, corticocancellous, osteochondral or vascularized), or locality of the graft (i.e. orthotopic [bone location] or heterotopic [non-bone location).Use: The cancellous bone is most commonly used in horses. It is very osteogenic and also has a potent osteoinductive capacity. Only 10 to 30% of the cells survive and only those on the surface of the graft. In horses, the cancellous bone can be harvested from the tuber coxae, sternum, and proximal tibia. Cortical bone graft is used uncommonly in horses or as part of a cortico-cancellous graft where it can contribute to the volume.The vascular response to a cancellous autograft is rapid, and the entire cancellous bed may be completely revascularized within 1 - 2 weeks. The cell population of this environment is predominantly osteoblasts, likely derived from the recipient and descendants of cells transplanted with the graft itself. Osteoblasts line the trabeculae of the graft and deposit a seam of osteoid that surrounds and entraps the grafted dead bone that is eventually resorbed by osteoclasts. Cortical grafts are revascularized less quickly than cancellous grafts because the dense cortical bone does not allow a large contact area for vascular penetration between the graft and the host. Instead, revascularization occurs via the old haversian and Volkmann canals, and then follows the process of creeping substitution, wherein the cortical bone graft is first resorbed before it is replaced with new viable bone.Drawbacks of bone graft: Unfortunately, bone grafts have drawbacks. The additional anesthetic time or personnel needed for graft harvesting and the potential for an insufficient quantity of graft, limited access to donor sites, loss of osteogenic cells, donor site infection, pain or hemorrhage, and failure of the donor bone are factors complicating cancellous autograft procedures. Similarly, allografts and xenografts – though less commonly performed in horses - carry the hazards of immune-mediated rejection and graft sequestration and, although unreported, the potential risk of disease transmission between donor and host. Bone banks are also costly to maintain.Bone graft substitutesAccordingly, bone-graft substitutes have been developed as alternatives to autologous or allogeneic bone grafts. In their simplest form, they consist of scaffolds made of synthetic or natural biomaterials that promote the migration, proliferation, and differentiation of bone cells for bone regeneration. They are generally more effective, however, when used as delivery vehicles for one or more Bone Growth and Differentiation Factors (BGDFs). They can be categorized into organic and inorganic matrices.Organic matricesOrganic matrices are comprised of two groups: Biological matrices such as demineralized bone matrix (DBM), non-collagenous proteins, collagen, fibrin and autolyzed antigen extracted allogeneic bone (AAA- bone) and synthetic matrices like polylactic acid or polyglycolic acid homo- or heteropolymers. Collagen and synthetic polymer delivery vehicles offer the greatest potential for clinical use at this time.Other organic delivery vehicles include hydrogels, autolyzed antigen-extracted allogeneic bone, inactivated dentin matrix, lyophilized cartilage and fibrin. They all suffer from poor load-bearing capacity and/or propensity for prompting foreign body responses. Table 2: Function of bone grafts and synthetic bone replacements Allographs, such as demineralised bone matrix, are possible in horses. However, they require special preparation (demineralisation) and storage. This is not practical in many equine orthopaedic situations, such as fractures where prompt treatment is crucial. Furthermore, the allogenic bone demonstrates a lower osteogenic capacity (lower rate of new bone formation), a higher resorption rate, a greater immunogenic response, and less revascularisation of the graft than the autogenous cancellous bone. In a rib-defect model in horses there was no evidence of enhanced healing associated with the use of equine demineralized bone matrix (Kawcak et al. 2000). These are some of the reasons why the allogenic bone has never become very popular in equine surgery.Limited availability and concerns relating to complications such as possible transmission of virus to the recipient have prompted search for bone-graft substitutes, such as ceramics (Calcium sulphate, Tricalcium phosphate, Hydroxyapatite) or single purified molecules for stimulating bone healing (bone morphogenic proteins, parathyroid hormone) or a combination of both. In equine surgery there is always the question of whether the cost of artificial bone replacements can be justified, because there is not enough clinical evidence for their efficiency. However a recent study revealed encouraging results in this regard (Perrier et al. 2008).So far, Gene-Therapy and Stem-Cell have not had a breakthrough, but Bone-Tissue engineering seems to be the rising star in this concert.Bone-tissue engineering combines progenitor cells, such as MSCs (native or expanded) or mature cells (for osteogenesis) seeded in biocompatible scaffolds and ideally in three-dimensional tissue-like structures (for osteoconduction and vascular ingrowth), with appropriate growth factors (for osteoinduction), in order to generate and maintain bone. The need for such improved composite grafts is obvious, especially for the management of large bone defects, for which the requirements for grafting material are substantial. At present, composite grafts that are available include bone synthetic or bioabsorbable scaffolds seeded with bone-marrow aspirate or growth factors (BMPs). Recently, an animal study has shown the potential for prefabrication of vascularized bioartificial bone grafts in vivo using b-TCP scaffolds intraoperatively filled with autogenous bone marrow for cell loading, and implanted into the latissimus dorsi muscle for potential application at a later stage for segmental bone reconstruction, introducing the principles of bone transplantation with minimal donor-site morbidity and no quantity restrictions. Another current approach to enhance bone regeneration and soft-tissue healing simultaneously is by local application of growth factors in the form of platelet-rich plasma (PRP), a volume of the plasma fraction of autologous blood with platelet concentrations above baseline, which is rich in many of the aforementioned molecules. A carrier matrix can be used (e.g. ACS). The PRP composite can then be wrapped around implants or the fracture itself or can be laid directly on the tissue bed of open wounds.

Tendon injuries often threaten the athletic career of performance horses. There may be single overloading but frequently the injury is the result of a gradual matrix degradation, initially without clinical signs, which may lead to impaired regenerative capacity. Another complicating factor is the lack of uniform pathology; frequently multiple stages of matrix integrity can be found and, therefore, there is no cure-all treatment. Ultrasound Tissue Characterization (UTC) is designed for tomographic visualization and quantification of 3-D matrix integrity. UTC is based on standardized compilation of ultrasound-data by means of an ultrasound probe that moves automatically along the tendon’s long axis, collecting transverse images every 0.2 mm, generating a 3-D volume. UTC-algorithms can discriminate 4 different echo-types, related to size and integrity of structures in the matrix (van Schie et al. 2003): Echo-type I, generated by intact and aligned fascicles with axial diameter ≥ spatial resolution. Echo-type II, generated by discontinuous, waving and/or swollen fascicles with axial diameter ≥ spatial resolution. Echo-type III, generated by a matrix mainly consisting of fibrils with axial diameter < spatial resolution. Echo-type IV, generated by a mainly amorphous matrix and fluid. Fundamental research with isolated tendons revealed that the ratios of these 4 echo-types are highly correlated with tendon matrix integrity, showing the discriminative power of UTC for tissue characterization (van Schie et al. 2009).Normal superficial digital flexor tendons in young mature horses are characterized by 80-90% type I, 10-15% type II and barely any type III and/or IV echoes. Loss of integrity is characterized by significant changes like decrease of type I, increase of type II (remodeling or inferior repair) and increase of type III (fibrillar) and/or IV (amorphous). Intra- and inter- observer reliability appeared to have intra-class correlations (ICC) ranging 0.92-0.98, indicative for excellent reproducibility. Clinical research revealed that UTC is sensitive and reliable to: • Monitor load-effects and detect matrix degradation (Plevin et al. 2019). Stage lesions for selection of appropriate intervention. • Quantify regenerative processes for objective evaluation of therapy and guided rehabilitation (Bosch et al. 2011).

Musculoskeletal regenerative medicine (RM) aims at restoring components of the musculoskeletal system. Most outcome parameters that are used to judge the efficacy of RM treatments focus on the degree to which the newly generated tissue resembles the native tissue and make use of histological, biochemical, and biomechanical techniques. However, in the end it is the functional outcome that counts, and gait is the functional product of the musculoskeletal system. In human medicine, measurement of function is based on the feedback of patients, mostly in the form of so-called visual analogue scoring (VAS) scales, which are seen as relatively reliable, though still subjective and susceptible to bias. In animals this is not possible, and gait is typically scored through application of semi-quantitative scores, like the American Association of Equine Practitioners (AAEP) scale in the horse. Various techniques to measure gait using parameters describing exerted forces (kinetic parameters) and parameters describing motion in space (kinematic parameters) have been developed over the past decades and in more recent times the use of some of these quantitative techniques has become standard in clinical practice in various places (Serra Bragança et al. 2018). It is expected that this use will rapidly increase further, which will have a profound impact on clinical practice. The current developments have already led to a discussion whether we should redefine lameness in the era of quantitative gait analysis (van Weeren et al. 2017). As an evaluation tool for measuring the functionality of musculoskeletal regenerative techniques, quantitative gait analysis has many advantages as it is an excellent technique for longitudinal monitoring in an objective and unbiased way and for subsequent documenting. In this field, the horse is leading but similar techniques are being developed for the dog. Future developments include the generation of big data sets and their exploitation for the development of pattern recognition techniques based on an artificial intelligence approach.

A fundamental base of bioengineering and tissue regeneration is the selection and development of the scaffolds responsible for cell growth. However, finding the “ideal” scaffold depends not only on proposing an innovative idea, but also on considering multiple chemical, biological, and physical aspects that can be manipulated to optimize their future clinical performance. Multiple local variables (such as local inflammation, vascularity, tissue damage, immune response, among others), as well as systemic variables (diseases or concomitant treatments) can favor or affect the scaffold behavior in each case. The selection of the ideal scaffold for each case involves three indispensable steps: design, selection of manufacturing material, and visualization of the future biological function that each biomaterial will perform. The design is always a parallel process with the selection of the ideal biomaterial. Certain “light” scaffolds (such as membranes, hydrogels, or sponges) will require the use of polymers that allow their simple manipulation and early degradation, which in turn can favor the release of charged molecules previously included, obtaining an active scaffold known as drug delivery system. On the other hand, structural scaffolds that are prone to replace block compromised structures may need different designs and production techniques, where three-dimensional printing is included. All of these options should consider important aspects such as bioactivity, regenerative capacity, and biological response of the surrounding tissues. Some alternatives may induce greater cell adhesion and proliferation, while optimizing the osseointegration and healing processes. Other alternatives may play a more “active” role while promoting regeneration processes and controlling local infectious diseases or painful responses. In order to look for the best translational approach of the biomaterial, each option must be chosen with the correct diagnosis of the case to be treated.

Tendons can be injured through over-strain at a number of different sites. When injured outside a synovial cavity (extra-thecal), injuries frequently repair by fibrosis, but this tissue is functionally deficient compared to normal tendon. Stem cells offer the prospect of improving this repair to restore function and enable a successful restoration of activity while minimizing the risk of re-injury. Naturally occurring equine Superficial Digital Flexor Tendon (SDFT) overstrain injuries usually have a contained lesion, thereby enabling simple intra-tendinous injection and, by the time of stem cell implantation, is filled with granulation tissue which acts as a vascularized scaffold. An anabolic drive is provided by mechanical loading of the tendon and the suspension of mesenchymal stem cells (MSCs) in bone marrow supernatant, which we have shown to have significant anabolic effects in vitro. To test the hypothesis that stem cells will enhance tendon healing, a controlled experimental study of naturally occurring SDFT injuries (n=12) has been performed (Smith et al. 2013). MSC treatment appeared to ‘normalize’ the tissue parameters so that they were closer to the contralateral, relatively normal, and untreated tendons than saline-injected controls, in spite of labelling experiments showing the majority of cells being lost within 24 hours (Becerra et al. 2013; Sole et al. 2013). A second adequately powered and independently analyzed study evaluated the clinical outcome of naturally occurring SDFT injuries treated using this technique (n=113), which showed a significantly reduced re-injury rate (Godwin et al. 2012). Intrasynovial (intra-thecal) tendon tears usually communicate with the synovial cavity where the synovial environment is particularly challenging for successful repair. However, MSCs administered intra-synovially failed to improve healing in either equine (naturally occurring) and ovine (induced) deep digital flexor tendon (DDFT) tears (Khan et al. 2018). Labelling of the implanted cells showed them to lodge within the synovium with no cells present in the tendon defect. Scaffolds are likely to offer better advantages for enhancing repair of intra-thecal tendon tears.

Bone is one of the few tissues capable of authentic regenerative repair. However, despite advances in surgical technique, orthopaedic hardware and our understanding of fracture biology, inadequate bone repair remains a major concern in both veterinary and human medicine. Cell-based technologies provide opportunities to utilize the osteogenic capacities of Mesenchymal Stem Cells (MSC) to augment bone repair. Much of the research on MSC biology has focused on cells derived from the bone marrow/endosteal compartment; however, osteoprogenitor cells (OPC) also reside in the periosteum. Periosteum develops as a fibro-cellular envelope surrounding developing skeletal elements. The inner, or cambium layer of periosteum, includes committed OPCs directly adjacent the bone surface, and a distinct sub-population of progenitors within the periosteal mid-substance that retain both chondrogenic and osteogenic capacities. During skeletogenesis, periosteal OPCs are responsible for appositional intramembranous bone formation that increases the radial diameter of long bones. Of critical importance, periosteal stem cells are the predominant cell population responsible for generating the cartilaginous or ‘soft’ callus that provides intermediate stabilization and a scaffold for subsequent callus ossification by endochondral ossification; the primary mechanism of bone repair. In recent experiments using isolates from ‘donor-matched’ periosteum and bone marrow, we have found that the basal osteogenic capacity of equine OPCs is considerably less than that of bone marrow-derived MSCs. Periosteal OPCs require exogenous Bone Morphogenetic Protein (BMP) for robust osteogenesis, a finding consistent with the clinical responses of bone to recombinant BMP protein. Perhaps more surprising, the osteogenic capacity of adult (2-10 years of age) OPCs is comparable to those of young foals’, although the cell yield is considerably greater from foal specimens. In light of the vital importance of callus formation for successful fracture healing of most, further research on the biology and clinical manipulation of periosteal OPCs is highly warranted.

Stem cell therapies hold promise for the treatment of musculoskeletal disorders. Mesenchymal stem cells (MSCs) derived from adult tissues are the most common type of stem cells being investigated for biomedical applications among all stem cell types. However, studies have shown that MSC properties and functions are largely affected by age and health condition of the donor, which often causes inconsistency in therapeutic outcomes. This is a critical challenge that needs to be addressed before the promise of stem cells for therapies can be fulfilled. Our group has worked on tackling the challenge for more than a decade by developing strategies such as priming the cell with regulatory molecules or hypoxia culture. Recently, we successfully reprogramed human and pig somatic cells into induced pluripotent stem cells (iPSCs) using the integration-free episomal method and subsequently derived MSCs from iPSCs for evaluation of potential orthopedic applications. Our study results showed that through cellular reprogramming the capacity of cell propagation and multilineage differentiation of MSCs was greatly enhanced and the expression of aging-associated markers in the cell was significantly downregulated, suggesting that cellular reprogramming can rejuvenate MSCs to increase the regenerative capability, and our approach converting MSCs into iPSCs is promising for addressing the challenge of reduced therapeutic potential associated with MSC aging. In addition, we found that during chondrogenic induction reprogramed MSCs increasingly differentiated into hyaline chondrocytes expressing cartilage-specific markers, compared to control parental cells, suggesting that iPSC-derived MSCs are promising therapeutic agents for articular cartilage regeneration. In general, our findings highlight the potential of iPSCs in better understanding aging-associated musculoskeletal disorders and providing biological options for the treatment.

Equine tendinopathy arises through two main mechanisms – external trauma or overstrain injury. The pathogenesis of the former is straight forward and prevention relies on avoiding risk factors for palmar/plantar lacerations and protecting the tendons through the use of boots. For over-strain injuries, these mostly arise from overloading of the distal limb resulting in mid-substance tearing of the digital flexor tendons or borders tears of the deep digital flexor tendon within the confines of the digital sheath and navicular bursa. While some of these injuries may be spontaneous injuries associated solely with overload (such as the intra-thecal injuries of the deep digital flexor tendon), it is widely accepted that most overstrain injuries of the superficial digital flexor tendon (and suspensory ligament) occur as a result of accumulated microdamage which predisposes the tendon to over-strain injury. The mechanisms of this accumulated microdamage are poorly understood but probably relate to the effect of high impact loading of the tendon, sustained during normal exercise, which drives degradative changes in the tendon fascicles (Dudhia et al. 2007) and, in particular, the interfascicular matrix (endotenon) that allows the fascicles to slide past one another as a mechanism for the spring-like extension of the tendon under load (Thorpe et al. 2013). This is compounded by the lack of adaptive remodelling in adult tendon (Smith et al. 2002). This subclinical damage makes the tendon prone to sudden tearing of the tendon matrix during normal exercise, the risk of which is increased by factors such as the firmness of the ground, weight, speed, and fatigue. Strategies for prevention of injury rely on identifying at risk individuals through more sensitive monitoring of tendon health, maximising the quality of tendon during growth using carefully tailored ‘conditioning’ exercise regimes (Smith & Goodship 2008), reducing the degeneration induced by normal training and competition, and avoiding high risk factors for the initiation of the clinical injury.

There is great interest to develop new materials with properties that resemble those of biological systems such as hierarchical organization, the capacity to grow or self-heal, and the ability to guide complex biological processes. These kinds of materials would open opportunities to engineer tissues with a much-needed higher level of complexity and overcome major obstacles in regenerative medicine. To this end, supramolecular chemistry offers an exciting opportunity to grow such materials from the bottom-up using molecules and processes found in nature. However, the ability to transform molecular and nano-scale design into functional devices with practical utility at the macroscale remains a challenge.The paper will describe new strategies that integrate supramolecular chemistry with engineering principles to develop practical materials with tuneable and advanced properties such as hierarchical organization, the capacity to grow, tuneable mechanical properties, and specific bioactivity (Inostroza-Brito et al. 2015; Aguilar et al. 2017; Elsharkawy et al. 2018; Hedegaard et al. 2018). These materials are being used towards new regenerative therapies of tissues such as enamel, bone, and blood vessels as well as creating more biologically relevant in vitro models.

Se obtuvieron muestras de sangre de 478 animales en siete hatos lecheros del Valle Central de Costa Rica, donde se sospechaba la presencia de Leucosis Vírica Bovina (L. V.B.). Sobre cada muestra se realizó un hemograma clasificando los animales en: positivos, sospechosos y negativos, según las claves de Gotze, Gottingen y por la morfología de los linfocitos. También se realizó la prueba de inmunodifusión en agar gel (G.I.D.). El método hematológi,co se comparó con el método serológico de inmunodifusión en agar gel (G.I.D.). Además se estudió la relación epidemiológica entre madres e hijas, así como la prevalencia de la enfermedad por edad. En uno de los hatos muestreados, se encontró una prevalencia de 39% por G.I.D., 23% por la clave de Gotze, 26°10 por la clave de Gottingen y 34% por la morfología de los linfocitos. Se corroboró la existencia de la Leucosis Vírica Bovina en Costa Rica, presentándose ensu forma enzoótica. La prueba de inmunodifusión en agar gel demostró ser un método más exacto, económico y rápido que las clásicas pruebas hematológicas en el diagnóstico de la Leucosis Vírica Bovina.