Biological injection therapy with leukocyte-poor platelet-rich plasma induces cellular alterations, enhancement of lubricin, and inflammatory downregulation in vivo in human knees: A controlled, prospective human clinical trial based on mass spectrometry imaging analysis.

Objective: To investigate the in vivo biological effects of leukocyte-poor platelet-rich plasma (LpPRP) treatment in human synovial layer to establish the cellular basis for a prolonged clinical improvement. Methods: Synovial tissues (n = 367) were prospectively collected from patients undergoing arthroscopic surgery. Autologous-conditioned plasma, LpPRP, was injected into the knees of 163 patients 1-7 days before surgery to reduce operative trauma and inflammation, and to induce the onset of regeneration. A total of 204 patients did not receive any injection. All samples were analyzed by mass spectrometry imaging. Data analysis was evaluated by clustering, classification, and investigation of predictive peptides. Peptide identification was done by tandem mass spectrometry and database matching. Results: Data analysis revealed two major clusters belonging to LpPRP-treated (LpPRP-1) and untreated (LpPRP-0) patients. Classification analysis showed a discrimination accuracy of 82%-90%. We identified discriminating peptides for CD45 and CD29 receptors (receptor-type tyrosine-protein phosphatase C and integrin beta 1), indicating an enhancement of musculoskeletal stem cells, as well as an enhancement of lubricin, collagen alpha-1-(I) chain, and interleukin-receptor-17-E, dampening the inflammatory reaction in the LpPRP-1 group following LpPRP injection. Conclusions: We could demonstrate for the first time that injection therapy using "autologic-conditioned biologics" may lead to cellular changes in the synovial membrane that might explain the reported prolonged beneficial clinical effects. Here, we show in vivo cellular changes, possibly based on muscular skeletal stem cell alterations, in the synovial layer. The gliding capacities of joints might be improved by enhancing of lubricin, anti-inflammation by activation of interleukin-17 receptor E, and reduction of the inflammatory process by blocking interleukin-17.

Magnetic resonance imaging and clinically controlled improvement of a combined autologous conditioned plasma combined with rh collagen type I injections in lateral epicondylitis.

The purpose of the study was to investigate the effect of combined autologous conditioned plasma and recombinant human collagen type I injections on lateral epicondylitis. Outcome was measured in 5 patients before the single application of ACP+rhCollagen type I (Arthrex ACP® Tendo) and after 10.60±3.58wks by means of (i) the Visual Analogue Scale for pain, (ii) range of motion for wrist extension/flexion as well as supination/pronation, and (iii) MRI-scans. VAS-scores significantly decreased from 6.40±1.14 at baseline to 1.80±2.49 at follow- up, and the effect was very large (p=0.04, dz =2.22). In addition, range of motion either improved or remained unrestricted, and MRI-scans showed healing of the extensor carpi radialis brevis tendon in most cases. A combined ACP+rhCollageninjection successfully reduces pain in lateral epicondylitis. Due to the small sample size, however, these promising preliminary results need further investigation in future research.

Positive effects of low level laser therapy (LLLT) on Bouchard's and Heberden's osteoarthritis.

BACKGROUND AND OBJECTIVE: Osteoarthritis (OA) is a common chronic disorder. While research usually focuses on OA of the large joints, OA of the hand receives relatively little attention resulting in a lack of a therapeutic gold standard. Low level laser therapy (LLLT)/photobiomodulation therapy has been successfully used to treat a variety of medical conditions. Nevertheless, its merits in the treatment of (hand) OA remain controversial. The aim of the present study was to examine the longitudinal effect of LLLT on the three major hand OA symptoms-pain, swelling, reduced joint mobility-in patients suffering from Bouchard's and Heberden's OA. STUDY DESIGN/MATERIALS AND METHODS: Thirty-four patients (32 females) aged 61.21 ± 2.13 years were administered 5-10 LLLT sessions to 85 joints (47 proximal and 38 distal interphalangeal joints). Therapy took place twice a week. Pain (Visual Analogue Scale), ring size (perimeter in mm), and range of motion (extension/flexion) were measured at baseline and after five treatments for all patients, and additionally after seven sessions and 8 weeks after treatment ended for patients who received more than five and seven treatments, respectively. Eighteen patients (37 joints) received only five treatments, 10 patients (29 joints) were administered seven treatments, and six patients (19 joints) were administered 10 LLLT sessions. RESULTS: LLLT significantly reduced pain and ring size and increased range of motion after five and seven treatments (all P's < 0.001). The effects were very large (all η(2) 's > 0.14). No further significant change occurred between 7 and 10 treatments. The effects achieved after seven sessions persisted for 8 weeks. CONCLUSIONS: LLLT is a safe, non-invasive, efficient and efficacious means to reduce pain and swelling and to increase joint mobility in patients suffering from Heberden's and Bourchard's OA. Further randomized controlled studies are needed to examine medium- to long-term effects as well as the ideal LLLT parameters. Lasers Surg. Med. 48:498-504, 2016. © 2016 Wiley Periodicals, Inc.

A new treatment for hip osteoarthritis: clinical evidence for the efficacy of autologous conditioned serum.

The purpose of the present study was to investigate the effect of intra-articular injections of autologous conditioned serum on human hip osteoarthritis and to test whether a potential treatment effect might be increased by additional injections of steroids and the recombinant interleukin-1 receptor antagonist protein anakinra. We compared the effects of autologous conditioned serum 46 hip osteoarthritis patients), autologous conditioned serum+cortisone (56 patients), and autologous conditioned serum+cortisone+recombinant interleukin-1 receptor antagonist protein (17 patients) in a retrospective clinical study by means of the Visual Analogue Scale for pain (pre- vs posttreatment). Over 14 months, treatment resulted in a large, statistically significant improvement for patients in all three groups, independent of the severity of osteoarthritis. Neither cortisone nor cortisone+recombinant interleukin-1 receptor antagonist protein increased the beneficial treatment effect over and above the effect of autologous conditioned serum alone. Autologous conditioned serum successfully reduces pain in hip osteoarthritis. In severe hip osteoarthritis, the sole application of autologous conditioned serum can be even more beneficial than the combination of autologous conditioned serum with steroids.

The use of recombinant human bone morphogenetic protein-2 for the treatment of a delayed union following femoral neck open-wedge osteotomy.

Although the clinical potential of bone morphogenetic proteins (BMPs) has been known for decades, their use in humans has only been approved for a limited number of orthopaedic conditions. Promising results in animals demonstrate the utility of BMP-2 in regional bone repair without using osteoconductors. To our knowledge, no comparable human case has been described. We report the case of a 50-year-old who suffered a femoral neck fracture. After 9 months of extensive treatment, he was still not pain-free. The following open-wedge osteotomy resulted in a therapy-resistant delayed union. We therefore conducted 4 computer tomography-guided injections of recombinant human (rh) BMP-2 into the bone gap. No osteoconductor was employed. Six weeks later, there was a 55-60% defect filling. Follow-up examination showed a complete union of the bone defect. Our case report shows that in a complicated delayed union rhBMP-2 can be successfully used to induce bone formation without any osteoconductor.

Clinical responses to gene therapy in joints of two subjects with rheumatoid arthritis.

This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1beta. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.

Bone-cartilage transplantation from the ipsilateral knee for chondral lesions of the talus.

PURPOSE: We present a prospective analysis to review talus dome chondral and osteochondral lesions treated with autogenous bone-cartilage transplantation harvested from the ipsilateral knee since 1998. The clinical outcome of osteochondral defects is investigated by using a method for resurfacing that supplies hyaline cartilage. The outcome analysis also considers defect size and the number of transplanted osteochondral cylinders. TYPE OF STUDY: Prospective analysis of a case series. METHODS: Included in the study were 43 patients with ankle joint pain resulting from osteochondritis dissecans stage III-IV (n = 22), post-traumatic cartilage defects (n = 16), and focal osteoarthritis (n = 5). The mean age of this group was 31.2 years; there were 30 male and 13 female patients. To carry out the osteochondral resurfacing procedure, anteromedial or anterolateral arthrotomy (23 cases) or medial malleolar osteotomy (20 cases) of the distal tibia was performed. The osteochondral autograft transfer system (OATS; Arthrex, Naples, FL) was used for transplantation. The follow-up examinations were performed after 3 months (clinical, radiological), after 6 months (clinical, radiological), after 9 months (clinical, radiological, hardware removal, and second-look arthroscopy), after 12 months, and every following year (clinical, radiological, magnetic resonance imaging). The follow-up of 11 patients was greater than 2 years (maximum, 4.5 years), for 8 patients 1 to 2 years, for 12 patients 6 to 12 months, and for another 12 patients 0 to 6 months. The results have been validated by the scores of Evanski and Waugh score and Mazur et al. RESULTS: The mean pain intensity measured by visual analogue scale (0 to 10, with 10 representing the worst imaginable pain) reduced from 4.4 to 2.3 after 6 months (n = 34), to 1.6 after 1 year (n = 23), and after 2 years to 1.1 (n = 14). Patients reported a significantly improved range of motion of the ankle compared with their preoperative status. The smaller the diameter of the transplants and the smaller the number of transplants used, the better were the results in pain reduction and postoperative range of motion. The Evanski and Waugh score improved from 52 to 88 points and the score described by Mazur et al. from 53 to 90 of 100 possible points. All medial osteotomies were healed clinically and radiographically. All grafts showed bony integration in the talus as seen in the radiographs and by magnetic resonance imaging. Second-look arthroscopy found integration of the osteocartilaginous graft with surrounding cartilage within the first year. A series of needle biopsies showed hyaline structure. CONCLUSIONS: Autogenous osteochondral transplantation of the talus using ipsilateral knee osteochondral grafts is a very promising surgical procedure to treat local cartilage lesions of the ankle joint. LEVEL OF EVIDENCE: Level IV.

Immunotherapy by gene transfer with plasmids encoding IL-12/IL-18 is superior to IL-23/IL-18 gene transfer in a rat osteosarcoma model.

BACKGROUND: Osteosarcomas are primary malignant tumors of bone or soft parts arising from bone-forming mesenchymal cells. Despite dramatic therapeutic advances, namely neo-adjuvant and adjuvant chemotherapy, progress is at a plateau. Cytokine-mediated gene therapy might represent a further advance in the therapy of the osteosarcoma. MATERIALS AND METHODS: We transfected UMR 108 osteosarcoma cells with different plasmids encoding IL-12, IL-23, proIL-18 and ICE (Interleukin-converting enzyme). IFN-gamma induction, which is known to induce antitumor effects mediated by the immune system, and cytotoxic effects of various cytokine combination were investigated. RESULTS: Our results show that local secretion of IL-12 by UMR 108 cells led to an induction of cytotoxic effects mediated by mononuclear cells, which were enhanced by additional administration of recombinant IL-18. In contrast to IL-18, IL-23 showed a moderate increase of IFN-gamma induction when transfected alone and could only slightly increase the IFN-gamma induction mediated by IL-12. IL-18 enhanced IFN-gamma induction when applied alone and was able to increase the IFN-gamma production that was induced by IL-12. CONCLUSION: IL-23 seems to be a less effective immuno-therapeutic for adjuvant treatment of osteosarcomas than IL-12 and IL-18, when taking only IFN-gamma induction into consideration.

Feasibility of percutaneous gene transfer to an atrophic nonunion in a rabbit.

Treatment of atrophic nonunions is a challenge to orthopaedic surgeons. Growth factors potentially are valuable factors for improvement of tissue healing. The use of growth factors, however, is limited by their short half-lives. Gene therapy has the potential to improve the treatment. This study aimed to establish and validate an atrophic nonunion model in a rabbit for the use of a percutaneous in vivo gene therapy protocol. An atrophic tibial nonunion was established in 24 New Zealand White rabbits. Radiologic and histologic followup was for 64 weeks. The rabbit tibias showed no radiologic or histologic signs of healing. In addition, an adenoviral vector carrying a marker gene was injected percutaneously into the nonunion site in 12 rabbits. Expression of the marker gene was assessed for as many as 4 weeks. The percutaneous gene delivery resulted in transgene expression in the nonunion site for as many as 4 weeks. The described model reliably leads to an atrophic tibial nonunion in rabbits. Adenoviral percutaneous gene delivery into the nonunion site is feasible and leads to transgene expression locally for at least 1 month. This study provides investigators with a reliable and reproducible model of an atrophic nonunion.

Gene transfer to the tendon-bone insertion site.

This study investigated whether gene transfer to the tendon-bone insertion site is possible during early tendon-transplant healing using viral vectors. In addition, we evaluated the optimal gene delivery technique for an in vivo adenoviral gene transfer to a tendon-bone insertion site in a bone tunnel. Twenty-six rabbits underwent a bilateral transfer of the flexor digitorum longus tendon into a bone canal in the calcaneus. The animals were divided into two groups. The first group (n=18) received a direct injection of an adenoviral vector carrying the luciferase marker gene into the tendon on the left side, while on the right side the adenoviral vector was first injected into the bone trough and the tendon was later inserted into the trough. The analysis of this experiment showed that over a 4-week period a higher luciferase activity was achieved using the bone trough immersion technique. In the second group (n=8) we therefore used the qualitative marker virus (Ad/-LacZ) with the bone trough immersion technique in order to show the site of gene expression. The histological analysis of this experiment demonstrated the presence of beta-galactosidase positive cells within the tendon-bone interface over a 4-week period. Therefore we showed in the first part of this study that the bone canal provides a more efficient target for direct adenoviral gene delivery than the tendon. In the second part of the study we demonstrated the feasibility of the bone trough immersion technique since sustained gene expression within the tendon-bone interface was obtained for up to 4 weeks. This study has shown the feasibility of gene delivery to the tendon-bone interface and provides the basis for the application adenoviral delivery of growth factor genes to the tendon-bone insertion site.

Isolated peripheral neurilemoma attached to the tendon of the flexor digitorum longus muscle.

Neurilemomas are benign tumours arising from peripheral nerves with a Schwann cell sheath. They are normally painless and slowly growing, rarely causing motor disturbances. Neurilemomas are most common in the cranial nerves, in the trunk, the upper and lower extremities, but may appear anywhere. Especially rare are neurilemomas of the lateral peroneal nerve in the region of the fibular head and in the foot. We present the first detailed report of a neurilemoma localized between the Achilles tendon and the flexor digitorum longus muscle with separation of the tumour from the tibial nerve.

Interleukin-12 and interleukin-18 induce indoleamine 2,3-dioxygenase (IDO) activity in human osteosarcoma cell lines independently from interferon-gamma.

PURPOSE: In this study we evaluated the ability of inducing activation of the enzyme indoleamine 2,3-dioxygenase (IDO) for the development of a new adjuvant therapy of osteosarcomas. The pathway that has described in the literature states that IDO activity is elevated by IFN-gamma. This mechanism is important because the increased IDO activation induces an intracellular degradation of the essential amino acid tryptophan and thereby activates apoptosis in human osteosarcoma cells. MATERIALS AND METHODS: Four different well-established human osteosarcoma cell lines (MNNG/HOS, KHOS-240, HOS and MG-63) were investigated in vitro. Several cytokines were tested for their ability to induce IDO activity. However special emphasis was placed to evaluate the synergistic effects of Interleukin-12 (IL-12) in combination with Interleukin-18 (IL-18). In the first series of experiments IDO induction was investigated by direct application of the cytokines IFN-gamma, TNF-alpha, IL-12 and IL-18 in different concentrations. Secondly, the increase of IDO expression from osteosarcoma cell lines was analysed in the presence of activated lymphocytes with or without cytokine application. RESULTS: Our results demonstrated that the combined application of IL-12 and IL-18 enhanced IDO activity in the human osteosarcoma cell lines HOS and MG-63, in the presence of activated lymphocytes. In the absence of activated lymphocytes, no significant enhancement could be detected. In all our experiments the increase in IDO expression was only partly inhibited by blocking INF-gamma. CONCLUSION: The presented study demonstrates that IL-12 and IL-18, or even more a combined application of both cytokines, induce IDO expression besides the known pathway via IFN-gamma. These mechanisms have been shown herein for the first time in human osteosacoma cell lines. Since IDO expression could still be shown after complete blocking of IFN-gamma, we conclude that at least a second pathway is responsible for inducing IDO activity. This is in contrast to the present knowledge about IDO activation.

Interleukin-12 and interleukin-18 change ICAM-I expression, and enhance natural killer cell mediated cytolysis of human osteosarcoma cells.

Cytokines play important roles in the expression of adhesion molecules and the function of anti-tumor effector cells in the immune system. In this study, the influence of interleukin-12 (IL-12) and IL-18 on the expression of ICAM-1 and natural killer (NK)-cell mediated lysis in a human osteosarcoma cell line (HOS) was evaluated. ICAM-I expression of HOS cells were analyzed by flow cytometry following treatment with IL-12, IL-18 or both, and in co-cultures with peripheral lymphocytes. NK-cell activation in response to IL-12 and IL-18 was investigated by selective flow cytometry using propidium iodide. ICAM-1 expression on HOS cells was significantly enhanced by IL-12, but only when co-cultured in cell-to-cell contact with peripheral lymphocytes. Antibodies to interferon-gamma abrogated this effect. If HOS cells and peripheral lymphocytes were separated in co-cultures, IL-18 could substitute for cell-to-cell contact, facilitating IL-12-mediated enhancement of ICAM-1. Addition of IL-18 also enhanced NK-mediated cytolysis of HOS cells. These findings demonstrate that IL-12 can enhance the expression of ICAM-1 in the presence of IFN-gamma and, with IL-18, enhances NK anti-tumor activity. Immunomodulation via cytokine therapy may lead to improved eradication of chemotherapy-resistant osteosarcomas.

rIL-18 triggered gene therapy based on a transduction with the IL-12 plasmid: a new option as immuno-therapy for osteosarcoma?

Gene therapy is a promising new method to treat tumors locally. Immuno-therapy for treatment of osteosarcomas is one option for hopefully improving the survival rate of patients with this tumor. Transduction of OS cells with the pCMV-IL-12neo plasmid induced a significant increase in IFN-gamma expression by mononuclear cells. This is known to induce antitumor effects mediated by the immune system. In combination with an administration of rIL-18, the IFN-gamma increase was multiplied in a dose-dependent manner. This study demonstrated that osteosarcoma cells can be targeted effectively in vitro by plasmids encoding the IL-12 gene. Considering the synergistic pathways it is reasonable to combine a local, gene transfer based on IL-12 with a rIL-18 administration to trigger the potentially promising immuno-effects for adjuvant treatment of osteosarcomas.