Evaluation of hematology, general serum biochemistry, bone turnover markers and bone marrow cytology in a glucocorticoid treated ovariectomized sheep model for osteoporosis research.

Osteoporosis is a metabolic disorder characterized by a loss of bone mass and structure and increasing the risk of fragility fractures, mostly among postmenopausal women. Sheep is a recognized large animal model for osteoporosis research. An experimental group of ewes (3-4 years old) was subjected to ovariectomy (OVX) and weekly glucocorticoid (GC) application for 24 weeks and compared with a sham control group. Blood and bone marrow parameters were analyzed before and 24 weeks after OVX and GC administration. Osteopenia was confirmed through micro-computed tomography and histomorphometric analysis of L4 vertebra in the study end. A statistically significant increase was observed in mean corpuscular volume, mean cell hemoglobin and monocytes and a decrease in red blood count and eosinophils (p<0.05). Alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, magnesium and α1-globulin increased, and creatinine, albumin, sodium and estradiol decreased (p<0.05). A slight decrease of bone formation markers (bone ALP and osteocalcin) and an increase of bone resorption markers (C-terminal telopeptides of collagen type 1 and tartrate-resistant acid phosphatase) were observed, but without statistical significance. This study aims to contribute to better knowledge of sheep as a model for osteoporosis research and the consequences that a performed induction protocol may impose on organic metabolism.

Preclinical and Translational Studies in Small Ruminants (Sheep and Goat) as Models for Osteoporosis Research.

PURPOSE OF THE REVIEW: This review summarizes research on the use of sheep and goats as large animal models of human osteoporosis for preclinical and translational studies. RECENT FINDINGS: The most frequent osteoporotic sheep model used is the ovariectomized sheep with 12 months post-operatively or more and the combined treatment of ovariectomized sheep associated to calcium/vitamin D-deficient diet and glucocorticoid applications for 6 months, but other methods are also described, like pinealectomy or hypothalamic-pituitary disconnection in ovariectomized sheep. The goat model for osteoporosis research has been used in a very limited number of studies in osteoporosis research relative to sheep. These osteoporotic small ruminant models are applied for biomaterial research, bone augmentation, efficacy of implant fixation, fragility fracture-healing process improvement, or bone-defect repair studies in the osteopenic or osteoporotic bone. Sheep are a recognized large animal model for preclinical and translational studies in osteoporosis research and the goat to a lesser extent. Recently, the pathophysiological mechanism underlying induction of osteoporosis in glucocorticoid-treated ovariectomized aged sheep was clarified, being similar to what occurs in postmenopausal women with glucocorticoid-induced osteoporosis. It was also concluded that the receptor activator of NF-κB ligand was stimulated in the late progressive phase of the osteoporosis induced by steroids in sheep. The knowledge of the pathophysiological mechanisms at the cellular and molecular levels of the induction of osteoporosis in small ruminants, if identical to humans, will allow in the future, the use of these animal models with greater confidence in the preclinical and translational studies for osteoporosis research.

Large Animal Models for Osteochondral Regeneration.

Namely, in the last two decades, large animal models - small ruminants (sheep and goats), pigs, dogs and horses - have been used to study the physiopathology and to develop new therapeutic procedures to treat human clinical osteoarthritis. For that purpose, cartilage and/or osteochondral defects are generally performed in the stifle joint of selected large animal models at the condylar and trochlear femoral areas where spontaneous regeneration should be excluded. Experimental animal care and protection legislation and guideline documents of the US Food and Drug Administration, the American Society for Testing and Materials and the International Cartilage Repair Society should be followed, and also the specificities of the animal species used for these studies must be taken into account, such as the cartilage thickness of the selected defect localization, the defined cartilage critical size defect and the joint anatomy in view of the post-operative techniques to be performed to evaluate the chondral/osteochondral repair. In particular, in the articular cartilage regeneration and repair studies with animal models, the subchondral bone plate should always be taken into consideration. Pilot studies for chondral and osteochondral bone tissue engineering could apply short observational periods for evaluation of the cartilage regeneration up to 12 weeks post-operatively, but generally a 6- to 12-month follow-up period is used for these types of studies.

Tartrate-resistant acid phosphate as biomarker of bone turnover over the lifespan and different physiologic stages in sheep.

BACKGROUND: Currently, the best resources for assessment of bone tissue using imaging techniques are expensive and available in few medical facilities, thus serum or urinary bone turnover biomarkers could be useful as early indicators of prognosis. However, there is a wide range of variability in bone turnover markers due to several factors, such as different ages and metabolic stages, thus it is important to have as much data published on the subject as possible. The aim of this study was therefore to generate a reference range for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) and validate the already published data. RESULTS: Serum alkaline phosphatase decreased with age, with statistical difference between the 1 month old and the other groups and between the over 8 years and the 6 months old groups. There was also a statistical difference in the ALP levels between the 3 to 5 years old gestation and lactation groups. For serum tartrate-resistant acid phosphatase, there was a statistical difference mainly between the 1 month old and the 6 months old, 6-8 years old, and above 8 years old groups. CONCLUSIONS: The results obtained could represent a useful tool for future studies using sheep as an animal model for orthopedic research. The different groups presented a wide variation of serum ALP and TRAP levels, however, these variations are entirely explained by known physiology. Therefore, this detailed study confirms the prediction that unexplained changes in these bone turnover markers do not occur during the lifespan of sheep.

Bone turnover markers in sheep and goat: A review of the scientific literature.

Bone turnover markers (BTMs) are product of bone cell activity and are generally divided in bone formation and bone resorption markers. The purpose of this review was to structure the available information on the use of BTMs in studies on small ruminants, especially for monitoring their variations related to diet, exercise, gestation and metabolic lactation state, circadian and seasonal variations, and also during skeletal growth. Pre-clinical and translational studies using BTMs with sheep and goats as animal models in orthopaedic research studies to help in the evaluation of the fracture healing process and osteoporosis research are also described in this review. The available information from the reviewed studies was systematically organized in order to highlight the most promising BTMs in small ruminant research, as well as provide a wide view of the use of sheep and goat as animal models in orthopaedic research, type of markers and commercial assay kits with cross-reactivity in sheep and goat, method of sample and storage of serum and urine for bone turnover markers determination and the usefulness and limitations of bone turnover markers in the different studies, therefore an effective tool for researchers that seek answers to different questions while using BTMs in small ruminants.

Bone turnover markers for early detection of fracture healing disturbances: A review of the scientific literature.

Imaging techniques are the standard method for assessment of fracture healing processes. However, these methods are perhaps not entirely reliable for early detection of complications, the most frequent of these being delayed union and non-union. A prompt diagnosis of such disorders could prevent prolonged patient distress and disability. Efforts should be directed towards the development of new technologies for improving accuracy in diagnosing complications following bone fractures. The variation in the levels of bone turnover markers (BTMs) have been assessed with regard to there ability to predict impaired fracture healing at an early stage, nevertheless the conclusions of some studies are not consensual. In this article the authors have revised the potential of BTMs as early predictors of prognosis in adult patients presenting traumatic bone fractures but who did not suffer from osteopenia or postmenopausal osteoporosis. The available information from the different studies performed in this field was systematized in order to highlight the most promising BTMs for the assessment of fracture healing outcome.

Serum total and bone alkaline phosphatase levels and their correlation with serum minerals over the lifespan of sheep.

This study aimed to assess serum total alkaline phosphatase (ALP) and its bone isoform (BALP) levels during the ageing and in different physiologic states of sheep, in order to expand the knowledge about the variation of these biomarkers over the sheep lifespan. Ninety female sheep were divided into nine groups of various ages and physiological states (dry, lactation and pregnancy). Serum ALP, BALP and mineral levels were determined by commercial immunoassay, molecular absorbance spectrophotometry and chemical luminescence for BALP determination. Serum ALP and BALP decreased as sheep aged, and no statistically significant differences were obtained between ewes in different physiologic states. The continuous decline of serum BALP concentration along the sheep lifespan, namely in mature and old sheep, is a sign of decreasing bone turnover associated with ageing. Serum calcium concentrations increased slightly until 2 years of age and then showed a tenuous but statistically significant decrease in mature sheep, while serum phosphorus maintained an uninterrupted decrease as sheep matured. The knowledge of serum values of bone biomarkers throughout the sheep lifespan may be useful in preclinical orthopaedic research studies and for animal science studies using sheep.

Management of canine wounds using platelet-rich fibrin (PRF) biomaterial. A case series report.

BACKGROUND: The increasing interest in platelet-based therapies has underwritten the development of novel veterinary regenerative treatments. The haemoderivative platelet-rich fibrin (PRF) comprises abundant concentrations of platelets and leucocytes, above the physiologic baseline, which are considered essential elements for wound regeneration, stimulating local angiogenesis, cellular migration, proliferation and differentiation, considered essential for skin repair. OBJECTIVES: This study aimed to describe the treatment of eight dogs with naturally occurring cutaneous wounds, where autologous PRF therapy was applied, using a protocol developed by our group. METHODS: Eight dogs, aged between 7-month and 9-year old, from different breeds and sexes, were enrolled in this study. Four of these wounds were clinically infected. In three cases, two PRF treatments were performed during the first week of treatment, followed by single weekly treatments from the second week onwards, until exophytic granulation tissues were present. In each case, the treatment was finalized only when complete wound closure was achieved. Wounds did not receive topical antiseptics, antibiotics or topical drugs to promote wound healing during the treatment. RESULTS: PRF-grafting treatments were well tolerated in all treated wounds, inducing significant granulation tissue formation. PRF clots acted as a natural tissue filler, promoting epithelization and wound closure, without the requirement of topical antimicrobial/antiseptics application, or additional surgical debridement. Evident skin contraction was recorded in larger injuries and all the treatments resulted in vestigial aesthetic scars where hair growth was also observed. CONCLUSIONS: PRF-therapy obtained promising results, as an alternative wound treatment, revealing a biological regenerative action, prompting the natural skin healing process.

Antinociceptive Effect of a Sacro-Coccygeal Epidural of Morphine and Lidocaine in Cats Undergoing Ovariohysterectomy.

BACKGROUND: A commonly described analgesic protocol for ovariohysterectomy (OHE) combines systemic opioids, sedatives, and non-steroidal anti-inflammatory drugs. However, systemic analgesia does not fully prevent perioperative visceral and somatic pain triggered by the surgical stimulus. OBJECTIVES: To compare the analgesic effects and quality of recovery of systemic analgesia with those of a sacrococcygeal epidural injection of lidocaine and morphine in cats undergoing elective OHE. Methods: Twenty domestic female cats were premedicated with dexmedetomidine (0.01 mg kg-1 IM) and alfaxalone (1.5 mg kg-1 IM) and randomly assigned to one of two analgesic protocols: methadone (0.2 mg kg-1 IM) in the control group CTR (n = 10) and methadone (0.1 mg kg-1 IM) + epidural (lidocaine 2% (0.3 mL kg-1) + morphine 1% (0.1 mg kg-1) diluted with NaCl 0.9% to a total volume of 1.5 mL in the SCC-E group (n = 10). General anaesthesia was induced with alfaxalone (1 mg kg-1 IV) and maintained with sevoflurane in 100% oxygen. Non-invasive blood arterial pressure and cardiorespiratory variables were recorded. The quality of recovery was assessed using a simple descriptive scale. Before surgery and 1, 2, 3, 4, 6, and 8 h post-op pain was assessed using the UNESP-Botucatu multidimensional composite pain scale (MCPS) and mechanical nociception thresholds (MNT). The repeated measures analysis of variance (ANOVA) was used to compare groups over time. Comparison between groups was performed using independent samples t-test if the assumption of normality was verified, or the Mann-Whitney test. The chi-square test of independence and exact Fisher's test were used to compare groups according to recovery quality. RESULTS: Heart rate and systolic arterial pressure increased significantly from baseline values in the CTR group and did not change in the SCC-E group. In the CTR group, MNT and UNESP-Botucatu-MCPS scores increased significantly from baseline for all assessment points and the first 3 h, respectively, whereas this did not occur in the SCC-E group. CONCLUSIONS AND CLINICAL RELEVANCE: Based on our results, the SCC-E administration of lidocaine 2% with morphine 1% is a reasonable option to provide perioperative analgesia in cats submitted to OHE, compared to a systemic protocol alone.

Mesenchymal Stem Cell Studies in the Goat Model for Biomedical Research-A Review of the Scientific Literature.

Mesenchymal stem cells (MSCs) are multipotent cells, defined by their ability to self-renew, while maintaining the capacity to differentiate into different cellular lineages, presumably from their own germinal layer. MSCs therapy is based on its anti-inflammatory, immunomodulatory, and regenerative potential. Firstly, they can differentiate into the target cell type, allowing them to regenerate the damaged area. Secondly, they have a great immunomodulatory capacity through paracrine effects (by secreting several cytokines and growth factors to adjacent cells) and by cell-to-cell contact, leading to vascularization, cellular proliferation in wounded tissues, and reducing inflammation. Currently, MSCs are being widely investigated for numerous tissue engineering and regenerative medicine applications. Appropriate animal models are crucial for the development and evaluation of regenerative medicine-based treatments and eventual treatments for debilitating diseases with the hope of application in upcoming human clinical trials. Here, we summarize the latest research focused on studying the biological and therapeutic potential of MSCs in the goat model, namely in the fields of orthopedics, dermatology, ophthalmology, dentistry, pneumology, cardiology, and urology fields.

Osteosynthesis Metal Plate System for Bone Fixation Using Bicortical Screws: Numerical-Experimental Characterization.

This study reports the numerical and experimental characterization of a standard immobilization system currently being used to treat simple oblique bone fractures of femoral diaphyses. The procedure focuses on the assessment of the mechanical behavior of a bone stabilized with a dynamic compression plate (DCP) in a neutralization function, associated to a lag screw, fastened with surgical screws. The non-linear behavior of cortical bone tissue was revealed through four-point bending tests, from which damage initiation and propagation occurred. Since screw loosening was visible during the loading process, damage parameters were measured experimentally in independent pull-out tests. A realistic numerical model of the DCP-femur setup was constructed, combining the evaluated damage parameters and contact pairs. A mixed-mode (I+II) trapezoidal damage law was employed to mimic the mechanical behavior of both the screw-bone interface and bone fractures. The numerical model replicated the global behavior observed experimentally, which was visible by the initial stiffness and the ability to preview the first loading peak, and bone crack satisfactorily.

Use of animal protein-free products for passaging adherent human adipose-derived stromal/stem cells.

Adherent adipose-derived stromal/stem cells (ASC) have been used in pre-clinical regenerative medical studies applied to a broad range of tissues with an ultimate goal of translating these findings to clinical safety and efficacy testing; however, many protocols passage the cells using porcine-derived trypsin. We have compared porcine trypsin with animal protein-free products from recombinant bacteria (TrypLE Express; Invitrogene) and corn (TrypZean; Sigma) based on cell yield, viability and immunophenotype. ASC harvested with each trypsin product were comparable.

The effect of storage time on adipose-derived stem cell recovery from human lipoaspirates.

Multipotent adipose-derived stromal/stem cells (ASCs) can be isolated with high yield from human subcutaneous lipoaspirates. This study reports our experience isolating, expanding, differentiating and immunophenotypically characterizing ASCs over a period of 4 days after having surgically obtained the lipoaspirate samples. The ultimate goal is to understand how to optimize the consistent isolation of ASCs from lipoaspirates. The length of time between adipose tissue harvest and processing will need to be systematically evaluated with respect to cell yield, viability, and function since some distance is likely to exist between the plastic surgeon's office where lipoaspiration is performed and the current Good Manufacturing Practices (cGMP) laboratory where the ASCs are isolated. The objective of this study was to determine the effect of time delays on the yield and function of ASCs after collagenase digestion. We were able to isolate ASCs from lipoaspirates up to 72 h after the surgical procedure. The ASCs isolated on sequential days after the original tissue harvest proliferated, differentiated and maintained cell surface markers. We found that the initial 24-hour period is optimal for isolating ASCs with respect to cell yield and that there was no significant difference between ASC cell proliferation and differentiation ability within this period of time. In contrast, each of these parameters declined significantly for tissues maintained at room temperature for 48 or 72 h prior to isolation. These findings should be considered in the future development of standard operating procedures for cGMP processing of clinical-grade human ASCs.

Canine-Origin Platelet-Rich Fibrin as an Effective Biomaterial for Wound Healing in Domestic Cats: A Preliminary Study.

Platelet-rich fibrin (PRF) is a recent platelet-based biomaterial, poised as an innovative regenerative strategy for the treatment of wounds from different etiologies. PRF is defined as a biodegradable scaffold containing elevated amounts of platelets and leukocytes having the capability to release high concentrations of bioactive structural proteins and acting as a temporal release healing hemoderivative. This study aimed to evaluate the performance of canine-origin PRF, obtained from blood of screened donors, as a regenerative biomaterial suitable for the treatment of critical wounds in felines. Four short-hair felines with naturally occurring wounds were enrolled in this study. Three of the wounds were considered infected. Each PRF treatment was the result of the grafting of newly produced PRFs at the recipient area. The PRF treatment was initially performed two to three times per week, followed by single weekly treatments. The study was finalized when complete wound closure was achieved. No topical antimicrobial/antiseptic treatment was applied. The present research demonstrated that xenogenic PRFs significantly induced healthy vascularized granulation tissue in lesions with soft tissue deficit, also prompting the epithelization at the injured site. No rejection, necrosis, or infection signs were recorded. Additionally, PRF-therapy was revealed to be a biological cost-effective treatment, accelerating the wound healing process.

Laterally applied single bone plate option for fixation of complete diaphyseal fracture of a third metatarsal bone in a circus work pony.

BACKGROUND: Complete fractures of the third metacarpal/metatarsal bones (Mc/t3) are not common, but can occur in various situations and all types of horses, contributing for approximately one third of all long bone fractures in the horse, mostly related to external trauma or to high energy injuries. To stabilize Mc/t3 fractures in the horse, conservative management with walking casts and/or open reduction and internal fixation techniques are referred in the scientific literature, these last one generally by double platting application to bone surface in 90 degrees. In the present case will be described a Mt3 complete diaphyseal fracture stabilization on an adult pony from a circus by applying only one bone plate. This work pony could return to its previous activity fully recovered 3 months after fracture stabilization. CASE DESCRIPTION: A 7-year-old, male, Shetland pony (103 kg), used for performance work at a circus was admitted to the Veterinary Teaching Hospital of University of Tras-os-Montes and Alto Douro, presenting on lateral recumbency after being struck by a truck. On physical examination, a penetrating wound of small dimension was detected at the medial aspect of the right hindlimb. After radiographic examination, a complete diaphyseal fracture with slight obliquity of the right Mt3 was confirmed. The owner opted for surgical repair thus fracture stabilization was achieved by the placement of just a single 8-hole 4.5 mm broad dynamic compression plate applied to the lateral face of Mt3 in compression function with 7 cortical screws (4.5 mm). Post-operatively a full limb cast was performed and maintained for 4 weeks, while the animal was kept under movement limitation at the hospital facilities for this period of time although the animal stayed until his full recovery 3 months' post-surgery. CONCLUSION: A complete recovery was achieved and the animal returned to the previous level of exercise without any significant postoperative complications or degree of lameness. In the authors knowledge, this is the first case report where stabilization of a complete Mt3 diaphyseal fracture was attain using a single compression plate contoured to the lateral face of the bone, instead of double plating, on an adult work pony from a circus subjected to a very demanding and intense physical activity.

Black-and-White Ruffed Lemur (Varecia variegata) in Captivity: Analysis of the Oral Microbiota in a One Health Perspective.

This study aimed to characterize the susceptibility profile to antibiotics and biofilm formation of Gram-negative bacterial isolates obtained from the oral cavity of the black-and-white ruffed lemur (Varecia variegata). From eight individuals from a zoo located in Portugal, samples of the oral microbiota were collected with sterile swabs and then placed in closed tubes with a transport medium. Culture was carried out for media of Gram-negative bacteria. Twenty-two isolates were obtained and subjected to susceptibility tests to twenty-five antimicrobial agents belonging to seven different classes. All tested isolates demonstrated resistance to, at least, one antibiotic, and it was possible to observe multidrug resistance in 11 of the 22 isolates (50%). It should be noted that an isolate showed phenotypic resistance to imipenem, an antibiotic for exclusive use in a hospital environment. All the isolates showed an increasing ability of biofilm formation over time. The obtained results show that wild mammals in captivity could be reservoirs and potential sources of multi-resistant pathogens. In view of this fact and considering the One Health concept, it will be advisable to establish local monitoring programs worldwide that benefit and protect human, animal and environmental health.

Clinical application of mesenchymal stem cells therapy in musculoskeletal injuries in dogs-a review of the scientific literature.

Mesenchymal stem cells (MSCs) are multipotent, which is defined by their ability to self-renew while maintaining the capacity to differentiate into a certain number of cells, presumably from their own germinal layer. MSCs therapy is based on their anti-inflammatory, immunomodulatory (immunosuppressive), and regenerative potential. This review aims to provide a clinical overview of the MSCs potential as a therapeutic option for orthopedic diseases in dogs. A total of 25 clinical studies published in the scientific literature in the last 15 years on various diseases will be presented: semitendinosus myopathy, supraspinatus tendinopathy, cruciate ligament rupture, bone fractures and defects, and also osteoarthritis (OA). All articles involved in this study include only diseases that have naturally occurred in canine patients. MSCs therapy in the veterinary orthopedic field has great potential, especially for OA. All studies presented promising results. However, MSCs bone healing capacity did not reveal such favorable outcomes in the long term. Besides, most of these clinical studies did not include immunohistochemistry, immunofluorescence, and histopathology to confirm that MSCs have differentiated and incorporated into the injured tissues. This review summarizes the current knowledge of canine MSCs biology, immunology, and clinical application in canine orthopedic diseases. Despite the positive results in its use, there is still a lack of defined protocols, heterogeneous samples, and concomitant medications used with MSCs therapy compromising therapeutic effects. Further studies are needed in the hope of overcoming its limitation in upcoming trials.

Bioengineered surgical repair of a chronic oronasal fistula in a cat using autologous platelet-rich fibrin and bone marrow with a tailored 3D printed implant.

Clinical summary: A tissue engineering approach was used to aid the surgical repair of a chronic oronasal fistula (ONF) in a 13-year-old cat. A three-dimensional (3D) printed mesh, tailored to the size and shape of the ONF, was created to support a soft tissue flap used to close the defect; and also to provide a matrix for mesenchymal stromal cells present in bone marrow aspirate and bioactive cytokines and growth factors present in platelet-rich fibrin harvested from the patient. A CT scan at day 75 after surgery revealed the formation of new tissue in the defect and the healing process was complete at follow-up 6 months after surgery. Relevance and novel information: Complications are frequently reported following surgical repair of ONFs and include dehiscence of the palatal suture line, flap necrosis due to damage to the greater palatine artery and maxillary osteomyelitis, mainly due to chronic infection and bone lysis. The case described here demonstrates how input from a multidisciplinary team and the use of a biomaterial, processed by sophisticated technologies, can create a precision regenerative medicine strategy adapted to the patient's clinical needs; this provided a novel therapeutic solution for an otherwise hard to treat clinical problem.

Evaluation of bone turnover markers and serum minerals variations for predicting fracture healing versus non-union processes in adult sheep as a model for orthopedic research.

Bone turnover markers (BTMs) have been considered as an auxiliary method of following the fracture healing process and for early prediction of impaired bone healing. A better understanding of the potential of BTMs in this application could allow for earlier interventions and improved patient care. The aim of this study with a large animal experimental model was to assess the variation of bone formation markers - namely the total alkaline phosphatase (ALP) and its bone-specific isoform (BALP), serum concentration of intact osteocalcin (OC), N-terminal propeptide type III procollagen (PIIINP) and of bone resorption markers - namely tartrate resistant acid phosphatase (TRAP) and deoxypyridinoline crosslink (DPD) during the first stages of a normal fracture healing process and of a segmental critical size defect (CSD), which progresses to a non-union process. Thirty healthy female sheep (Portuguese Churra-da-Terra-Quente breed), approximately 4-years-old, were enrolled in this study. Jugular venous blood samples were collected pre-operatively and at 1, 2, 3, 4, 6, 8, 10 and 12 post-operative weeks. The animals of the CSD group showed significant lower serum levels of BALP, OC and significant higher serum PIIINP levels at early stages of the fracture healing process, compared with animals that progressed in a normal fracture healing process. Serum BALP, OC and PIIINP levels could be useful as non-invasive auxiliary tools with other complementary methods for predicting the outcome of traumatic bone fractures.

Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells.

Recent studies suggest that bone marrow stromal cells are a potential source of osteoblasts and chondrocytes and can be used to regenerate damaged tissues using a tissue-engineering (TE) approach. However, these strategies require the use of an appropriate scaffold architecture that can support the formation de novo of either bone and cartilage tissue, or both, as in the case of osteochondral defects. The later has been attracting a great deal of attention since it is considered a difficult goal to achieve. This work consisted on developing novel hydroxyapatite/chitosan (HA/CS) bilayered scaffold by combining a sintering and a freeze-drying technique, and aims to show the potential of such type of scaffolds for being used in TE of osteochondral defects. The developed HA/CS bilayered scaffolds were characterized by Fourier transform infra-red spectroscopy, X-ray diffraction analysis, micro-computed tomography, and scanning electron microscopy (SEM). Additionally, the mechanical properties of HA/CS bilayered scaffolds were assessed under compression. In vitro tests were also carried out, in order to study the water-uptake and weight loss profile of the HA/CS bilayered scaffolds. This was done by means of soaking the scaffolds into a phosphate buffered saline for 1 up to 30 days. The intrinsic cytotoxicity of the HA scaffolds and HA/CS bilayered scaffolds extract fluids was investigated by carrying out a cellular viability assay (MTS test) using Mouse fibroblastic-like cells. Results have shown that materials do not exert any cytotoxic effect. Complementarily, in vitro (phase I) cell culture studies were carried out to evaluate the capacity of HA and CS layers to separately, support the growth and differentiation of goat marrow stromal cells (GBMCs) into osteoblasts and chondrocytes, respectively. Cell adhesion and morphology were analysed by SEM while the cell viability and proliferation were assessed by MTS test and DNA quantification. The chondrogenic differentiation of GBMCs was evaluated measuring the glucosaminoglycans synthesis. Data showed that GBMCs were able to adhere, proliferate and osteogenic differentiation was evaluated by alkaline phosphatase activity and immunocytochemistry assays after 14 days in osteogenic medium and into chondrocytes after 21 days in culture with chondrogenic medium. The obtained results concerning the physicochemical and biological properties of the developed HA/CS bilayered scaffolds, show that these constructs exhibit great potential for their use in TE strategies leading to the formation of adequate tissue substitutes for the regeneration of osteochondral defects.