The Influence of Temporary Epiphysiodesis of the Proximal End of the Tibia on the Shape of the Knee Joint in Children Treated for Leg Length Discrepancy.

Background: Leg length discrepancy (LLD) is a common problem in the daily clinical practice of pediatric orthopedists. Surgical treatment using LLD temporary epiphysiodesis with eight-plate implants is a minimally invasive, safe, and well-tolerated procedure that provides good treatment effects with a relatively low percentage of complications. The main aim of this retrospective study was to determine the effect of epiphysiodesis on the shape of the proximal tibia. Methods: The retrospective study was based on medical records from 2010 to 2019. Radiographs taken before the epiphysiodesis and at 6-month intervals until the end of the treatment were investigated. A total of 60 patients treated for LLD were included in the study (24 girls, 36 boys). They were divided into three groups depending on the duration of the LLD treatment: group I (18 months), group II (30 months), and group III (42 months of treatment). Radiological parameters were assessed, including the roof angle (D), the slope angles (α and ß), and the specific parameters of the tibial epiphysis, namely LTH (lateral tubercle height), MTH (medial tubercle height), and TW (tibial width). Results: The roof angle decreased in all the groups, which was accompanied by an increase in the ß or α angle. LTH, MTH and TW also increased, and the differences before and after the treatment for the treated legs were statistically significant in all the studied groups. The greatest change in the shape of the articular surface of the proximal tibia occurred after 42 months of treatment. Conclusions: The study showed that epiphysiodesis affects the proximal tibial articular surface over prolonged treatment. Thus, there is a need for future long-term follow-up studies to elucidate the potential effects of LLD egalization.

The activity of monocyte-derived macrophages after stimulation with platelet-rich and platelet-poor concentrates. Study on an ovine model of insertion of a tibial implant coated with silicon-doped diamond-like carbon.

Introduction: Macrophages are crucial immune cells that play a role in tissue repair and can exhibit pro- or anti-inflammatory behaviour based on environmental stimulation. Their functional phenotype can be affected by platelet-derived products as determined by those products' composition. When the inflammatory response caused by implantation is excessive, it can lead to rejection of the implant. Therefore, a thorough evaluation of implant haemocompatibility is necessary to minimise undesirable consequences. Material and Methods: In an in vitro study, monocyte-derived macrophages (MDMs) were obtained from the whole blood of sheep after a silicon-doped diamond-like carbon-coated implant insertion. These MDMs were then exposed to autologous platelet-derived products for functional marker analysis. Results: Platelet-poor plasma (PPP) and pure platelet-rich plasma (P-PRP) stimulation increased arginase-1 activity, while leukocyte-rich PRP stimulation produced a mixed response involving higher O2- (6.49 ± 2.43 nM vs non-stimulated 3.51 ± 1.23 nM, P-value < 0.05) and NO (3.28 ± 1.38 µM vs non-stimulated 2.55 ± 0.32µM, P-value < 0.05) generation. Conclusion: Using PPP and P-PRP stimulation in post-implantation procedures may contribute to the polarisation of macrophages towards the M2-like pro-resolving phenotype, thereby accelerating wound healing. This would also prevent implant degradation due to an excessive inflammatory process.

NiTi Shape Memory Clamps with Modified Surface for Bone Fracture Treatment.

Using NiTi alloys with shape memory for long-term medical implants requires modification of their surface due to the possible occurrence of corrosion. Hence, the surface of the staples used to join fractured bone within the craniofacial region was modified by applying a titanium oxy-nitrogen layer and a hydroxyapatite coating. Surface-modified clamps were tested in vivo using New Zealand white rabbits. After determining the mechanical characteristics of the bone and considering the initial state and surface modification, the diameter of the wire (used to make the clamps with the appropriate compression force) was selected. Implantation was performed on two groups of rabbits: experimental and control. In the experimental group, an intentionally induced bone fracture was treated in one tibia. On the second tibia, two additional clamps were applied to increase the possibility of a negative impact of the NiTi alloy on a living organism. After 6 weeks of application, a proper joining of the broken bone fragments was stated. Whereas after twelve weeks, no negative impact of the clamp material on a living organism, i.e., a rabbit, was found. Hence, the clamp with the modified surface can connect bone fragments in humans as well as small and medium-sized animals, with an extended range of use up to 12 weeks.

Reactivation of Vertebral Growth Plate Function in Vertebral Body Tethering in an Animal Model.

Flexible spine tethering is a relatively novel fusionless surgical technique that aims to correct scoliosis based on growth modulation due to the pressure exerted on the vertebral body epiphyseal growth plate. The correction occurs in two phases: immediate intraoperative and postoperative with growth. The aim of this study was to evaluate the reactivation of vertebral growth plate function after applying corrective forces. The rat tail model was used. Asymmetric compression and distraction of caudal growth plates were performed using a modified external fixation apparatus. Radiological and histopathological data were analysed. After three weeks of correction, the activity of the structures increased across the entire growth plate width, and the plate was thickened. The height of the hypertrophic layer and chondrocytes on the concave side doubled in height. The height of chondrocytes and the cartilage thickness on the concave and central sides after the correction did not differ statistically significantly from the control group. Initiation of the correction of scoliosis in the growing spine, with relief of the pressure on the growth plate, allows the return of the physiological activity of the growth cartilage and restoration of the deformed vertebral body.

Osteoarthritis: Pathogenesis, Animal Models, and New Regenerative Therapies.

Osteoarthritis (OA) is a chronic, progressive, multifactorial disease resulting in a progressive loss of articular cartilage structure and function that is most common in middle-aged and older patients. OA is involved in the loss of extracellular matrix and cartilage as well as cell number decreases within the matrix, especially in the further stages of the disease. The immune system plays a pivotal role in the pathomechanism of this condition. Both humoral and cellular mediators contribute to cartilage destruction, abnormal bone remodeling, synovitis, and joint effusion. The increasing prevalence of this disease has led to a growing interest in using animal models as the primary way to broaden the knowledge of the pathogenesis of OA and possible therapies at each stage of disease development. This review aims to describe the signs, pathogenesis, and classification of OA as well as discuss the advantages and disadvantages of some animal models. The currently used treatment methods include mesenchymal stem cells, exosomes, gene therapies, and blood-derived products. In addition, exogenous growth factors, platelet-rich plasma (PRP), platelet lysate, and autologous conditioned serum (ACS) are discussed with the application of tissue engineering techniques and biomaterials.

Effects of Topical Treatment of Foot Rot in Sheep Using Ozonated Olive Ointment.

INTRODUCTION: Foot rot in small ruminants is highly contagious, causes severe lameness, and impairs fertility and wool and meat production. It is usually treated with parenteral antibiotics, with attendant antibiotic resistance risk, and with bactericidal footbaths, potentially harmful to humans and the environment. An alternative treatment in sheep is proposed based on repeated topical ozonated ointment application. Its effectiveness and safety were evaluated by estimation of acute-phase response, biochemical indicators of organic damage, and antioxidant/oxidant balance (AOB). MATERIAL AND METHODS: The study was conducted on ten sheep with Egerton scale 2-3 lesions. Ozone application was repeated every day for seven days. Blood was drawn first (T0) after foot cleaning and before ozonation, then (T1) seven days after the first ozone application, and finally (T2) four days after the last application. RESULTS: High clinical effectiveness was observed, with total recovery by 28 days from the start of treatment. A significant increase in antiradical activity was noted on the basis of a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay from 1.16 ± 0.04 µmolTe/mL at T0 to 1.23 ± 0.03 µmolTe/mL at T1, with a slight decrease in oxidative stress. Calculated on the basis of antiradical capacity, AOB was higher at T1 (130 ± 19%) and decreased to 110 ± 16% at T2. Calculated on the basis of reducing power, it was 169 ± 22% at T1 and 131 ± 17% at T2. CONCLUSION: These results indicated that the AOB is efficient enough to prevent oxidative organ injury and the applied doses of ozone are safe for animals.

Structure and Pathologies of Articular Cartilage.

The aim of the review was to describe a complex microstructure and biomechanical properties of the articular cartilage as well as a current review of its pathologies encountered in veterinary practice. The articular cartilage with its unique features: complex microarchitecture, significant mechanical durability and elasticity, lacking blood, lymphatic vessels, and innervation, seems to stand in contradiction to the laws of biology. It can be involved in a vast majority of diseases, from osteoarthrosis as a result of natural aging process to more complex in nature like osteochondromatosis. The primary role of articular cartilage is to provide the surface for movement in any single joint in the body. Therefore, its diseases lead to physical impairment and deterioration of the quality of life. Treatment of articular cartilage poses a formidable challenge in both modern human and animal medicine.

Application of Natural Neutrophil Products for Stimulation of Monocyte-Derived Macrophages Obtained before and after Osteochondral or Bone Injury.

We evaluated the use of some neutrophil products, namely; autologous rabbit antimicrobial neutrophil extract (rANE), heterologous porcine antimicrobial neutrophil extract (pANE), neutrophil degranulation products (DGP) and neutrophil microvesicles (MVs) for stimulation of monocyte-derived macrophages (MDMs) to improve healing. Two animal models were evaluated; the rabbit model for autologous osteochondral transplantation (OT) with application of rabbit ANE, DGP or MVs for MDMs stimulation, and the ovine model of the insertion of a Ti implant with the use of porcine ANE, and ovine DGP or MVs for MDMs stimulation. Macrophage activity was assessed on the basis of free radical generation and arginase activity. We estimated that DGP acted in a pro-inflammatory way both on rabbit and ovine MDMs. On the other hand, MVs acted as anti-inflammatory stimulator on MDMs in both experiments. The response to ANE depended on origin of extract (autologous or heterologous). Macrophages from rabbits before and after OT stimulated with autologous extract generated lower amount of NO and superoxide, especially after transplantation. In the ovine model of Ti implant insertion, heterologous ANE evoked increased macrophage pro-inflammatory activity. Our study revealed that these neutrophil products could regulate activity of macrophages, polarizing them into pro-or anti-inflammatory phenotypes that could enhance bone and osteochondral tissue healing.

Prospects and Applications of Natural Blood-Derived Products in Regenerative Medicine.

Currently, there are a number of therapeutic schemes used for the treatment of various types of musculoskeletal disorders. However, despite the use of new treatment options, therapeutic failure remains common due to impaired and delayed healing, or implant rejection. Faced with this challenge, in recent years regenerative medicine started looking for alternative solutions that could additionally support tissue regeneration. This review aims to outline the functions and possible clinical applications of, and future hopes associated with, using autologous or heterologous products such as antimicrobial peptides (AMPs), microvesicles (MVs), and neutrophil degranulation products (DGP) obtained from circulating neutrophils. Moreover, different interactions between neutrophils and platelets are described. Certain products released from neutrophils are critical for interactions between different immune cells to ensure adequate tissue repair. By acting directly and indirectly on host cells, these neutrophil-derived products can modulate the body's inflammatory responses in various ways. The development of new formulations based on these products and their clinically proven success would give hope for significant progress in regenerative therapy in human and veterinary medicine.

In vivo behavior of biomicroconcretes based on α-tricalcium phosphate and hybrid hydroxyapatite/chitosan granules and sodium alginate.

The current studies provide insights into how predictions based on results of physicochemical and in vitro tests are consistent with the results of in vivo studies. The new biomicroconcrete type materials were obtained by mixing the solid phase, composed of hybrid hydroxyapatite/chitosan granules and highly reactive α-tricalcium phosphate powder, used as the setting agent. This approach guaranteed a good adhesion of the continuous cement phase to the surface of granules. It has been demonstrated that developed biomicroconcretes are surgically handy, possessed favorable physicochemical and biological properties and can be considered as effective bone implant material. The hierarchical porosity and compressive strength (2-6 MPa) similar to cancellous bone made them suitable for low-load bearing applications. Despite the fact that final setting times of biomicroconcretes were longer than recommended in the literature (i.e., exceeded 15 min), their short cohesion time allows for a successful implantation in a rabbit femoral defect model. Histological analysis and Raman studies revealed newly formed bone tissues around the sides of implanted materials. Furthermore, the process of neovascularization and reconstruction of the bone tissue, as well as a reverse scaffolding process, was visible. No signs of inflammation or adverse tissue reactions were observed during the experiment.

Changes in the Activity of Ovine Blood-derived Macrophages Stimulated with Antimicrobial Peptide Extract (AMP) or Platelet-rich Plasma (PRP).

INTRODUCTION: Antimicrobial peptides (AMP) are a large group of innate immune effectors, which apart from antimicrobial activity show immunomodulative properties. Platelet-rich plasma (PRP) is a source of autologous growth factors and is used for stimulation of bone and soft tissue healing. The purpose of this study was to assess the influence of PRP and AMP extract on ovine monocyte-derived macrophage cultures. MATERIAL AND METHODS: The study was conducted on ovine macrophages (Mfs) previously stimulated with LPS or dexamethasone and then with preparations of PRP or AMP. Following activation of the Mfs their morphological and functional features were assessed. RESULTS: The study revealed pro-inflammatory influence of both examined preparations on Mfs cultures on the basis of morphology, ROS generation and arginase activity. Both preparations enhanced the pro-inflammatory response of cultured Mfs. CONCLUSION: This activity may intensify the antimicrobial action of Mfs, however, in cases of excessive and prolonged inflammation the use of these preparations should be limited.

The Use of Allogenic Stromal Vascular Fraction (SVF) Cells in Degenerative Joint Disease of the Spine in Dogs.

BACKGROUND/AIM: Stem cells are widely used in regenerative medicine and in clinical practice for the treatment of damaged nerve tissue, myocytes, tendons, and ligaments. The aim of the study was to monitor VEGF levels after the administration of allogenic cellular material (SVF) in the course of treatment of dogs suffering from degenerative joint disease in the spinal region. MATERIALS AND METHODS: The study was conducted on 10 dogs of both genders, aged between 6 and 13 years in which allogenic stromal vascular fraction of stem cells (SVF) was administered intravenously. The control group was composed of 10 clinically healthy dogs. Before treatment and after 2- and 8-week intervals blood samples were obtained from the study group dogs in order to determine VEGF levels via immunoenzymatic test. RESULTS: in a few days after the therapy, alleviation of pain symptoms and reduction of lameness were noticed. The VEGF level in 2 weeks after the therapy was significantly elevated (median: 38.77 pg/ml), while in 8 weeks a decrease was observed (median: 18.37 pg/ml). Conlusion: Administration of allogenic stem cells has a positive influence on elevation of the VEGF levels in the blood serum of affected animals as well as their regeneration capacity.

Impact of a pulsed magnetic field on selected polymer implant materials.

PURPOSE: Physiotherapy with the use of pulsed magnetic fields is one of the methods of activating the processes of bone healing and regeneration. Exposing materials serving as membranes in guided bone regeneration (GBR) or guided tissue regeneration (GTR) to magnetic fields is an effective model that allows to monitor changes in the material under the influence of the magnetic field. METHODS: Materials engineering methods were used to verify the extent of material degradation resulting from magnetic field exposure in an aqueous environment. Changes in surface morphology were observed under an optical microscope and a scanning electron microscope (SEM). Changes in surface wettability were analysed in relation to the direct contact angle. Chemical structural changes were verified with the use of infrared spectroscopy (FTIR-ATR). RESULTS: The PCL-based membrane materials underwent relatively moderate surface degradation (altered contact angle, changes in surface morphology), but the absence of observable FTIR-ATR spectral shifts evidenced material stability under the influence of magnetic field. More extensive degradation processes were observed in the case of PLDLA-based materials, whose surface character changed from hydrophilic to hydrophobic. The spectra revealed enhanced intensity of the chain terminal groups, provided that modifiers (nanometric SiO2 and TCP (water reservoir)) were present in the polymer matrix. CONCLUSIONS: The extent degradation in the polymer membrane was primarily dependent on the presence of aqueous environment, while the influence of the magnetic field on the analysed membrane materials was negligible. Therefore, GBR/GTR membrane implants can be considered to remain stable during rehabilitation with the use of alternating magnetic field.

Allergic reaction to platelet-rich plasma (PRP): Case report.

RATIONALE: In the recent years, growing interest is focused on the use of platelet-rich plasma (PRP) in wound healing and tissue regeneration. There are a number of papers regarding the usefulness of PRP in the healing of ulcerations, skin injures, bone loss or distraction osteogenesis. Most authors emphasize the safety of PRP usage due to its authogenic nature. PATIENT CONCERNS: We present a case of a 14 -year-old boy admitted to our department due to simple bone cyst of the distal tibia, qualified for injection of PRP into the cyst. PRP was separated with the use of Magellan Autologous Platelet Separator System (Arteriocyte Medical Systems Hopkington, MA) according to the manufacturers' manual. Immediately after separation during short-term IV anaesthesia, 3 mL of PRP was installed to the bone cyst under image intensifier control. DIAGNOSES: Within the first 24 hours after exposure to PRP, the skin rash appeared. Physical examination revealed the small red papular, regionally purpuric eruptions, mainly concentrated on the upper extremities and on more warmed regions of skin, in association with pharyngitis, tonsillar enlargement, mucopurulent discharge in the posterior pharynx and swelling of the eyelids. INTERVENTIONS: As the patient received calcium citrate with the PRP injection additional calcium citrate test were performed. Skin prick testing (negative) was and an intradermal test was positive (10×13 mm). Treatment included Claritine (Loratidinum) and Clemastin (Clemastinum)-both antihistaminic drugs. OUTCOMES: All symptoms withdrew and the patient was released home after 4 days. The patient is in 6 years follow-up without any symptoms of allergic disease. LESSONS: Our case shows that safety of use of PRP is not absolutely sure. The pure autologous tissue is safe, but preparation for its use can substantially decrease this safety. In our patient, only limited skin reaction to calcium citrate was observed, but general reaction leading to anaphylactic shock cannot be excluded. In order to reduce the risk of side effects skin test should be performed but as there were no records of allergic diseases on family and patients medical history this should apply to all patients.

Treatment of Articular Cartilage Defects: Focus on Tissue Engineering.

The treatment of articular cartilage defects seems to be one of the greatest challenges in modern orthopaedics. From a medical point of view there are 3 main goals to achieve for cartilage trauma treatment: restoration of the joints motion, pain relief and elimination/delay of the onset of osteoarthritis. Treatment can be divided into conservative (including pharmacotherapy, arthrocentesis and physiotherapy) and surgical. The last comprises reparative techniques, regenerative methods and symptomatic treatment. While both are focused on reconstruction of the damaged cartilage, the difference lies in the type of filling tissue. Reparative techniques include: drilling of the subchondral bone, spongiolisation, abrasion, mictrofracture, and autologous matrix induced chondrogenesis (AMIC). Regenerative methods contain: periosteal and perichondral grafts, mosaicplasty, autologous chondrocyte implantation and matrix-induced autologous chondrocyte implantation (MACI). Nowadays tissue engineering, including gene therapy, is emerging as one of the key approaches to cartilage treatment and holds promises for new achievements and better outcomes of many cartilage diseases and traumas.

The Neutrophil Response to Rabbit Antimicrobial Extract After Implantation of Biomaterial into a Bone/Cartilage Defect.

BACKGROUND/AIM: In this study, the neutrophil response to an antimicrobial extract was evaluated as a potential marker of inflammatory process after implantation of alginate and carbon fiber biomaterials into a bone or cartilage defect in rabbits. MATERIALS AND METHODS: The response to biomaterials used was assessed based on enzyme release, generation of reactive oxygen species (ROS) and survival of neutrophils isolated from the rabbit's blood after implantation. RESULTS: The implantation of alginate biomaterial increased elastase and alkaline phosphatase release, whereas carbon fibers did not evoke increased elastase release. The implantation of both biomaterials resulted in a higher myeloperoxidase (MPO) release after 30-min incubation. Stimulation with different fractions of antimicrobial peptide (AMP) extract diminished MPO release and nitric oxide generation, as well as slightly reducing neutrophil survival. CONCLUSION: Our study permits the assessment of the neutrophil intravital response to an implant without the need for preparation of histological sections. Additionally, AMP extract restricted some manifestations of the pro-inflammatory response and may be considered a regulator of neutrophil activity in the early inflammatory phase, preventing rejection of the implant.

Application of Platelet-rich Plasma and Tricalcium Phosphate in the Treatment of Comminuted Fractures in Animals.

AIM: To assess the applicability of ß-tri-calcium phosphate (TCP) and platelet-rich plasma (PRP) in the treatment of comminuted fractures in small animals. MATERIAL AND METHODS: The experimental study was carried out on 16 New Zealand White rabbits. After creating the bone defect and performing tibial osteotomy, TCP implants containing activated PRP were introduced into the fracture and the defect. The fracture was stabilised using external fixators or intramedullary nails. After 12 weeks, the animals were euthanised, and radiological, histological, scanning electron microscopy and peripheral quantitative computed tomography examinations were performed. The analysis also covered the results of fracture treatment in 37 small animals (cats and dogs) in which treatment with TCP containing PRP was used as an alternative to cancellous bone implantation. RESULTS: Correct bone union was observed in the experimental groups, TCP remained visible at the site of the fracture after 12 weeks. In the clinical application in small animals, bone union was observed in over 91% of treated animals. CONCLUSION: ß-TCP and activated PRP may be an effective method of bone union enhancement in the treatment of comminuted fractures in small animals.

Elevated EGF Levels in the Blood Serum of Dogs with Periodontal Diseases and Oral Tumours.

BACKGROUND/AIM: Paradontopathy and neoplasms of the oral cavity represent one of the greatest challenges in human and animal dentistry. EGF plays a key role in maintaining the integrity and proper rate of cell proliferation in normal oral epithelium. The aim of the present study was to study serum levels of EGF in dogs diagnosed with periodontal diseases and oral cavity tumours. MATERIALS AND METHODS: The samples comprised of cancerous tissue sections and serum obtained from dogs of various breeds, aged between 5-13 years. Serum EGF concentrations were measured by an immunoenzymatic method. RESULTS: The median for EGF concentration in serum of dogs suffered from severe periodontal diseases was greater when compared to the control group. EGF concentration in dogs with malignant tumours was significantly higher than in those with non-malignant growths. A positive correlation between EGF concentration and tumour size was also observed. EGF level in dogs diagnosed with benign tumours was comparable to the control group. CONCLUSION: The blood serum level of EGF increases significantly in patients with malignant oral tumours and advanced periodontal disease. In malignant tumours, the high level of EGF correlates with the size and invasiveness of the neoplasm.

Osteochondral Repair Using Porous Three-dimensional Nanocomposite Scaffolds in a Rabbit Model.

AIM: To evaluate the utility of a novel nanocomposite biomaterial consisting of poly-L/D-lactide, and hydroxyapatite bioceramics, enriched with sodium alginate in articular cartilage defect treatment. MATERIALS AND METHODS: The biomaterial was prepared using the method of solvent casting and particle leaching. The study was conducted on 20 New Zealand White rabbits. Experimental osteochondral defects were created in the femoral trochlear grooves and filled with biomaterials. In control groups, the defects were left to spontaneously heal. The quality of newly-formed tissue was evaluated on the basis of macroscopic and histological assessment. Additionally the level of osteogenic and cartilage degradation markers were measured. RESULTS: The majority of the defects from the treatment group were covered with tissue similar in structure and colour to healthy cartilage, whereas in the control group, tissue was uneven, and not integrated into the surrounding cartilage. CONCLUSION: The results obtained validate the choice of biomaterial used in this study as well as the method of its application.

Different activation of monocyte-derived macrophages by antimicrobial peptides at a titanium tibial implantation in rabbits.

The aim of our study was to assess the functional and morphological features of monocyte derived Mfs from rabbits with titanium (Ti) tibial implants, their ability to polarize towards M1 or M2 and their reactivity after stimulation with neutrophil antimicrobial peptides extract (AMP). The study was conducted on six White New Zealand rabbits with tibial implants of pure titanium Grade 2. Blood was taken before implantation and 14days after biomaterial implantation. Then, blood-derived Mfs were cultured and their function was assessed on the basis of morphological changes, generation of nitric oxide (NO), superoxide, and proteases release after treatment with LPS or dexamethasone (Dex). As a result of polarization we specified two subpopulations of Mfs with features characteristic of each subtype. M1 stimulated with LPS exhibited an increased NO and superoxide level, and M2 stimulated with Dex which showed higher arginase and lower free radical generation. Addition of AMP evoked further changes in Mfs morphology and function. After 24h stimulation with AMP an increase of NO was observed in all cultures, whereas after 48h it decreased. Production of superoxide lowered, especially after 48h, when M1 generated 6.00±0.2nM and M1/AMP culture generated 5.2±0.1nM of superoxide. Our study revealed that activated Mfs stimulated with AMP demonstrated both pro- and anti-inflammatory features. Moreover, we did not detect significant differences between the response of Mfs cultured from blood derived monocytes before and after implantation of Ti implants.