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

The influence of porcine prophenin on neutrophils isolated from rabbit blood during implantation of calcium sulphate graft material into bone tissue

[EN] Immune dysfunction induced by surgical trauma may comprise either an inappropriately exaggerated inflammatory response or a profound suppression of cell- mediated immunity. Neutrophils are the leading cells in the first response to trauma. It is known that they mediate initial resistance to bacterial infection. Activated neutrophils can degranulate and release some enzymes such as elastase and myeloperoxidase (MPO). The function of elastase is, among others, to kill bacterial, whereas MPO is a specific enzyme of primary granules of neutrophils and a marker of in vivo neutrophil activation. Previous reports estimated that some cathelicidins could act to increase or diminish an innate immune response in which neutrophils participate. The aim of this study was to evaluate prophenins (PF) isolated from porcine leukocytes in respect to neutrophil activity and survival during implantation of calcium sulphate bone grafts substitution in rabbits. Obtained results pointed out that neutrophils responded to PF depending upon concentration. Thirty min from implantation of calcium sulphate graft, we observed the greatest release of elastase (57.01±0.49% of maximal release) in cultures stimulated with 10 mg/ml of PF, at 0 mg/ml was 51.15±0.23%, while after 24 h of incubation the greatest response was at a concentration of 20 mg/ml. MPO release after 30 min from surgery decreased significantly at 10 mg/ml. In higher concentrations, the inhibition was less pronounced. Moreover, we estimated that PF causes cytotoxicity in the highest concentration as well as the apoptosis of neutrophils.Wessely-Szponder, J.; Bobowiec, R.; Szponder, T. (2012). The influence of porcine prophenin on neutrophils isolated from rabbit blood during implantation of calcium sulphate graft material into bone tissue. World Rabbit Science. 20(3):163-172. doi:10.4995/wrs.2012.1149SWORD16317220

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

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

The influence of autologous and heterologous extract of antimicrobial peptides on leukocytes isolated during titanium implant insertion in rabbit and ovine model

This study evaluated the in vitro leukocyte response to titanium implants in the presence of autologous or heterologous antimicrobial peptides extracts. Antimicrobial peptides (AMPs) appeared to be a new approach both against microorganisms and for regulation of inflammatory and repair processes. To evaluate their potential usefulness in regenerative medicine, we prepared different extracts of neutrophil-derived AMPs from rabbit, ovine or porcine blood which contained AMPs of different compositions, mainly defensins, cathelicidins and fragments thereof. Then, we assessed in vitro the influence of different AMPs extracts on the neutrophils and monocyte-derived macrophages (MDM) activity. For this purpose, these cells were obtained from experimental animals, rabbits, or sheep submitted to insertion of a titanium implant into the tibial defect. The cultured cells stimulation was autologous or heterologous, dependently on the AMPs extract origin and the experimental animal species. The neutrophil activity was assessed on the basis of the enzymes release from azurophilic and secondary granules and the free radicals generation. The MDM functional assessment was based on the NO and superoxide generation and arginase activity. Additionally, morphological changes were evaluated in the cell cultures. Our results indicated that the origin of AMPs extract is crucial for its activity. The autologous extracts stimulated anti-inflammatory responses, whereas the heterologous extracts displayed pro-inflammatory effect on neutrophils and macrophages. These results might be considered during the introduction of new preparations in regenerative medicine

Impact of a pulsed magnetic field on selected polymer implant materials

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

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

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

Preparation and preliminary in vivo studies of resorbable polymer modified with allogenic bone chips for guided bone regeneration and orthopedic implants

Composites made of resorbable polylactide modified with bone powder are part of the current search for implantable materials endowed with advantageous biomechanical functions, which make them suitable for orthopedics and traumatology applications. The bone additive containing active bone morphogenetic proteins (BMPs) and calcium phosphates introduced into the polymer matrix is to grant the implant with a biological activity. Subsequently, the resorbable matrix should get replaced with bone tissue. In order to avoid losing the osteoinductive properties of the designed material, it should be processed at low temperatures via physicochemical methods. This paper is devoted to the preparation and optimization of the composite production method suitable for biodegradable polymers and morphogenetic proteins along with the assessment of biocompatibility and biological properties of obtained materials. The tape-casting method was successfully applied. Resorbable polymer (medical poly-L-lactide, Purasorb PL38 by Purac) with 15 wt% of human bone powder (from tissue bank) were used to fabricate PLA-CP/BMPs composite implants. They were tested in in vivo studies that were performed in rabbit bone tissues. The results show a high biocompatibility of the material and good osteointegration with bone tissue

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 β), 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 β 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