Purpose

Reactive arthritis (ReA) is known as aseptic arthritis secondary to extra-articular infection, but its pathogenesis is still unclear. We have reported that "the inflammation amplifier" in which NF-κB and STAT3 coactivation in non-immune cells cause chronic inflammation. Here, we hypothesized that post-infection exosomes play an important role in the development of ReA via the inflammation amplifier. The objects are to create a new ReA model and to elucidate the mechanism of ReA pathogenesis through the inflammation amplifier using the new model.

Methods and Materials

The chronic inflammation model mice (F759) were used to evaluate the arthritis development after salmonella administration. Intra-articular administration of serum exosomes after salmonella infection to non-infected mice was performed to evaluate the arthritis induction effect of the exosomes. To analyze the inflammation amplifier activation mechanism via exosomes from distant organs, CagA (cytotoxin-associated gene A) was used. CagA is pathogen of Helicobacter pylori and is known to affect distant organs via exosomes.

Results

Following salmonella infection, increment of serum IL-6, enhancement of IL-6 expression in the ankle joint and phosphorylation of STAT3 in the ankle joint were observed. Thus, it was clarified that digestive tract infection of salmonella caused the inflammation amplifier activation in distant organs, especially ankle joints. Furthermore, exacerbation of clinical score was confirmed by inducing micro-bleeding in ankle joints in addition to salmonella infection. This result indicated that the success of ReA model mice creation and suggested that pathogenic agents might exist in the blood. Intraarticular administration of serum exosomes from salmonella-infected mice to uninfected mice showed an increase in clinical score. CagA-containing exosomes resulted in activation of the inflammation amplifier and exacerbation of the clinical score.

Conclusion

We succeeded in creating a ReA model mouse with a high incidence rate. Involvement of the inflammation amplifier activation mechanism via serum exosome was suggested for the ReA development.

Purpose

There have been many reports mentioned about tissue regeneration efficacy of mesenchymal stem cells (MSCs) for treatment of osteoarthritis (OA). One of the main mechanism of transplanted MSCs is the trophic effect by secretome, which is represented to exosome. The aim of this study is to evaluate the suppression effect of tissue degenerating of exosome secreted by MSCs on the treatment of OA.

Methods and Materials

Exosomes (Exos) secreted by adipose tissue derived MSCs were extracted using an ultracentrifuge method from conditioned medium (serum-free medium). Nano tracking analysis and western blots were used to identify exosomes. Exosomes were injected in the collagenase-induced OA (CIOA) mouse model and histomorphometric analyses of joints were performed by μCT and Osteoarthritis Research Society International (OARSI) score.

Results

The majority of Exos were approximately 50–150 nm in diameter and expressed CD9, CD81. Macroscopically the structure of cartilage was degenerated and subchondral bone was exposed partially in untreated group (CIOA without Exos). In contrast, much of cartilage were remain in treated group (CIOA with Exos). The area of bone resorption at the distal site of femoral epiphyseal line evaluated by μCT was suppressed 65% in treated group compared with untreated (Figure 1). Moreover histological analysis of tibias sections indicated that cartilage tissues of treated mice presented almost typical hyaline features although with a little bit superficial fibrillation. However, untreated group showed typical degenerative OA changes including superficial zone delamination and proteoglycan depletion. OARSI score of treated mice improved compared to untreated (Figure 2).

Figure 1. The area of bone resorption at the distal site of femoral epiphyseal line evaluated by μCT.

Figure 2. Histological (OARSI) score at the section of tibial plateau.

Conclusion

These results were confirmed that exosome could protect cartilage degradation. Exosome may be the next generation therapeutics for OA instead of MSC therapy.

Purpose

Platelet-rich plasma (PRP) is commonly used in orthopaedic sports medicine. However, neither PRP preparation or clinical application (concentration, timing, number of PRP applications) are standardized, making research and clinical use challenging. Therefore the aim of this study is to investigate the effect of standardized PRP powder with different concentrations, timing and number of applications in a highly standardized cell culture model.

Methods and Materials

A characterized, lyophilized platelet growth factor preparation (PRP powder) was produced (Kieb et al. AJSM 2017) and used for standardized stimulation of human chondrocytes. Cells were cultivated over 2 weeks with different stimulation frequency (2x, 3x and 6x) and different concentrations of growth factor lyophilisates (0.5 %, 1 % and 5 %) in several cell culture medium combinations. Metabolic cell activity and cell proliferation were analyzed after 7 and 14 days. Phenotypic changes were visualized by live-dead-staining. Additionally, synthesis of bone-specific collagen type 1 and cartilage-specific collagen type 2 were quantified by ELISA, whereas sulfated proteoglycans and glycosaminoglycane were analyzed by Blyscan^TM Assay.

Results

Chondrocytes showed a significant dose- and time-depend increase in both metabolic cell activity and cell number. Also a significant increase of the bone-specific procollagen type 1 and GAGs were seen, whereas no significant differences in the cartilage-specific collagen type 2 were detected. Microscopically, cells changed their typical chondrogenic phenotype to a fibroblast-like phenotype during the cultivation.

Conclusion

Our results show a substantial potential of a standardized growth factor preparation in basic science research and future tissue regeneration. The stimulation of human chondrocytes by standardized growth factor preparations to induce or inhibit proliferation and differentiation of human cells is one very promising field of research in tissue engineering. Current problems with PRP such as absent standardization, lack of consistency among studies, and black box dosage could be solved by using characterized PRP powder made by pooling and lyophilizing multiple platelet concentrates.

Purpose

Autologous anti-inflammatories (AAI) are a class of blood-derived products with high concentrations of anti-inflammatory cytokines that are currently being investigated for the treatment of mild to moderate knee osteoarthritis (OA) to determine if they can ameliorate symptoms longer or better than traditional intra-articular injections such as hyaluronic acid (HA) and steroids. The purpose of this evaluation is to determine the duration of effect from a single injection of APS before patients elect a new treatment course.

Methods and Materials

Forty-six patients underwent a 2:1 randomization process to either one single injection of APS (n=31) or saline (n=15) (NCT02138890). APS was prepared with the nSTRIDE APS Kit (Zimmer Biomet). The 12 month double-blind outcomes of the double-blind portion of the trial were previously published. The APS cohort was asked to participate in unblinded long-term follow-up. Efficacy endpoints of pain and function over time (WOMAC LK 3.1, KOOS, and VAS) were measured as a change from baseline to each time point. Quality of life (SF-36), OMERACT-OARSI responder criteria, rescue medication usage, as well as yearly x-rays and 24 month MRI imaging were completed.

Results

Survivorship of the APS cohort that agreed to long-term follow-up was 71% at 3 years. At 36 months, the mean WOMAC Pain improvement was 70%(Figure 2)(7.8 ± 4.0) in the APS cohort, which was a significant improvement compared to the baseline score(p<0.0001). APS cohort patients also showed a statistically significant improvement in their KOOS pain score (114%,33.3 ± 22.0; p<0.0001) and VAS pain score (42.7%, 2.0 ± 3.3; p=0.0012).

Conclusion

Intra-articular injections of APS for mild to moderate knee OA was safe and a portion of patients continue to have pain relief 3 years after a single injection. Clinical investigation (NCT03182374) to determine the long-term efficacy compared to a single injection hyaluronic acid is currently ongoing.

Purpose

To investigate platelet-rich plasma (PRP) combined with gelatin sponge (GS) to improve tendon-bone interface healing and structure formation.

Methods and Materials

Characterization of the GS scaffold was performed with a scanning electron microscope, and the release curve after loading with PRP was evaluated. A real-time reverse transcription quantitative polymerase chain reaction assay was performed to test the levels of tendon-to-bone healingerelated gene expression. Finally, 18 New Zealand white rabbits were randomly divided into 3 groups and underwent semitendinosus autograft anterior cruciate ligament reconstruction: autograft group without PRP, PRP group, and PRP-GS group. All rabbits were killed 8 weeks after the operation. Magnetic resonance imaging scans, biomechanical testing, and histologic evaluation were performed.

Results

An enzyme-linked immunosorbent assay and CCK-8 assay showed that the GS could control the release of PRP and prolong its bioactivity time, as well as promote bone marrow mesenchymal stem cell proliferation. In the PRP-GS group, the levels of related genes were upregulated compared with the PRP group (P < .05). Lower signal in the magnetic resonance images indicated fibrocartilage formation in the 2 groups with PRP. In addition, histologic staining showed that the tendon-bone connection had a greater fibrocartilaginous transition region in the PRP-GS group, and the histologic scores were higher (vs the PRP group, P ¼ .039). The maximum failure load and stiffness were higher in the PRP-GS group than in the other 2 groups.

Conclusion

GS loading with PRP could prolong the bioactivity time of PRP and promote bone marrow mesenchymal stem cell proliferation and osteogenic gene expression in vitro. It also promoted the early healing process at the tendon-bone junction in a rabbit anterior cruciate ligament reconstruction model.

Purpose

To analyze the effect of a brief, 4-minute bout of high intensity interval exercise on the cellular and growth factor content of platelet-rich plasma

Methods and Materials

10 volunteers were recruited. 15mL of blood was harvested to create platelet-rich plasma (PRP) using the autologous conditioned plasma (ACP) system. Volunteers then performed 4 minutes of high intensity interval exercise (HIIE) on an exercise bike. After a brief recovery period, blood was re-drawn and a second PRP sample created.

Samples were analyzed for cellular content (platelets, leukocytes, red blood cells and mean platelet volume) and growth factor content (PDGF, VEGF, IGF and TGF).

Paired sample t-tests were used to determine mean differences. An a priori alpha level off 0.05 was used to determine significance. A post-hoc Bonferroni correction was applied to correct for family-wise error rate, changing the significance level to less than or equal to 0.006.

Results

Results

A significant difference in cell count between pre- and post-exercise PRP concentrations was noted only for platelet count. Mean platelet count was 367.4 ± 57.5 thousand per microliter at baseline and rose to 497.7 ± 93.3 thousand per microliter after the four minute exercise protocol. Mean values for the other three categories saw increases, but were not statistically significant.

A significant difference in growth factor count between pre- and post-exercise PRP concentrations was noted only for TGF. Mean TGF concentration was 8237.2 ± 7676.5 pg/mL at baseline and rose to 21535.7 ± 4062.6 pg/mL after the four minute exercise protocol. Mean values for the other three growth factors all saw increases, but none were statistically significant.

Conclusion

A brief, 4 minute bout of high intensity interval exercise increases the platelet and TGF content of PRP. Further clinical research is needed to determine if these increases result in superior clinical outcomes.

Purpose

The purpose of this study is to define the potential of platelet lysate as an animal-free alternative to FBS in expansion of human chondrocytes for cell therapy purposes. Additionally, the effects of using platelet lysate for redifferentiation of chondrocytes in 3D pellets are assessed.

Methods and Materials

Platelet lysate was prepared from human blood and platelet enrichment was measured. Chondrocytes were isolated from human osteoarthritic cartilage (age 55-84, mean 66 years) and subjected to a range of concentrations of platelet lysate in monolayer culture. Cell proliferation and morphology were assessed after 7 days. In addition, expression of chondrogenic genes was determined by quantitative real-time PCR. Next, platelet lysate-expanded chondrocytes were cultured in 3D cell pellets and cartilage matrix production was assessed after 28 days. Glycosaminoglycan (GAG) and collagen production in the pellets were quantified by biochemical analyses as well as (immuno)histochemistry. Besides, platelet lysate was supplemented into redifferentiation medium of chondrocytes in 3D pellets. Cartilage matrix production was assessed with similar outcomes.

Results

Platelet lysate had a dose-dependent effect on chondrocyte proliferation, but expression of chondrogenic markers was decreased in monolayers when compared to serum-expanded monolayers. GAG production after 28 days of subsequent 3D pellet culture was significantly higher in pellets consisting of chondrocytes expanded with 1% platelet lysate compared to controls (figure 1). When used for redifferentiation of chondrocyte pellets, platelet lysate significantly decreased the production of GAGs and collagen. This was confirmed by (immuno)histochemistry.

Conclusion

In conclusion, low concentrations of platelet lysate stimulate chondrocyte proliferation in 2D monolayer and cartilage ECM production in subsequent 3D pellet culture. However, this does not work for high concentrations of platelet lysate. Furthermore, this study showed unfavourable effects on redifferentiation of 3D chondrocyte cultures in platelet lysate. Thus, platelet lysate is a promising animal-free alternative to be used for chondrocyte expansion.

Purpose

Background: PRP has established its short term role in symptomatic relief in early OA knee. New variants and modification of PRP may give prolonged and better results

Null Hypothesis: Chitosan poloxamer gel with PRP (Chitosan PRP) is better than PRP in terms of anti-inflammatory effects.

Study Design: Controlled Laboratory Study

Methods and Materials

44 Dunkin Hartley guinea pigs were included (weighing ~ 700g). Ten animals (group DC) were randomized into disease control group where no intervention was done in either knees. Groups 1, 2 and 3 (Ten animals each) were given single intraarticular Injection of PRP, Chitosan gel alone and Chitosan+PRP in one knee respectively. Isotonic saline injection was given in the contralateral knees in groups 1,2 and 3 as control. Five animals from each group (subgroup DC.3, 1.3,2.3 and 3.3) were euthanized at three months and the remaining five (subgroup DC.6, 1.6, 2.6 and 3.6) at six months post intervention. Upon euthanasia, knee joint synovial fluid samples were collected from each joint for COMP estimation by ELISA and histologic assessment of articular cartilage and synovium was done.

Results

Mean synovitis scores was significantly lower in PRP and Chitosan PRP group compared to disease control and Chitosan gel group at 3 months (p < 0.05). No difference was noted at 6 months. However, there was no significant difference between Chitosan PRP and PRP groups at 3 or 6 months. There was no significant difference in total articular scores and mean synovial fluid COMP concentration among the groups at 3 or 6 months.

Conclusion

Single injection of PRP exerts an anti-inflammatory effect in the short term but not in the long term. Addition of chitosan poloxamer gel did not offer any additional advantage compared to PRP and hence Null hypothesis is rejected

Purpose

Articular cartilage lesions can be treated with bone marrow stimulating techniques in combination with three-dimensional scaffold implants (m-BMS) to support the chondrogenic differentiation of mesenchymal progenitor cells (MPC), which enter the defect after opening of the subchondral bone plate. The process of repair tissue formation is often supported by the use of autologous platelet-rich plasma (PRP) in an undefined preparation with unpredictable concentrations of growth and differentiation factors. The use of a scaffold combined with an allogeneic, off-the-shelf PRP could lead to a standardized, more practical therapy for clinical routine.

Methods and Materials

A migration assay was performed to determine the biological activity of allogeneic, lyophilized PRP produced by two manufacturers. Scaffold implants made of polyglycolic acid and hyaluronan (PGA-HA) were immersed with the two PRP-preparations, lyophilized, seeded with MPC and cultured in vitro. The chondrogenic effect of a single dose of lyophilized PRP was determined via real-time gene expression analysis of typical chondrogenic marker genes and immunohistochemical staining of extracellular cartilage-related matrix molecules.

Results

A single dose of both lyophilized PRP preparations induced the expression of cartilage-related marker genes and histochemical staining revealed a proteoglycan-deposition in both PRP groups after stimulation over 21 days. Migration assays confirmed a significant migratory effect of both PRP-preparations before and after lyophilization.

Conclusion

A single dose of allogeneic, lyophilized PRP from two standardized preparations is able to support cell migration, chondrogenic differentiation and subsequent matrix deposition in PGA-HA scaffolds. In addition, lyophilization has no negative impact on biological activity of PRP. Thus, it is possible to produce an easy-to-handle, off the shelf implant, whose cell stimulating effects could be favorable for a m-BMS therapy of cartilage defects.

Purpose

Background:Platelet Rich Plasma (PRP) has emerged as the forerunner amongst disease modifying treatment options of early osteoarthritis of knee. However there is no consensus on optimum dosing schedules.

Purpose: To determine whether multiple injections of PRP (Three) are better than single injection of PRP in a guinea pig knee OA model in short and long term.

Study Design: Controlled Laboratory Study

Methods and Materials

Methods:36 Dunkin Hartley guinea pigs (weighing ~600-800g) were chosen for this study. They were divided into group DC (disease control group), group G1 (single PRP group) and group G2(multiple PRP group) containing 10, 10 and 12 animals respectively. Four animals were used for preparation of allogeneic PRP. Groups G1 and G2 received one and three injections of PRP (at weekly interval) respectively. No intervention was done in group DC.Half of the animals from each group (subgroup DC.3, G1.3 and G2.3) were euthanized at three months and the remaining half (subgroup DC.6, G1.6 and G2.6) at six months post intervention. Histologic assessment for articular cartilage and synovium was done.

Results

Results: Mean synovial scores of single PRP group and multiple PRP group were significantly better than disease control group at 3 months. There was no difference between single and multiple PRP group at 3 months. At 6 months, the multiple PRP group was significantly better than single PRP and disease control groups in terms of mean synovial scores. The mean articular cartilage scores in multiple PRP group were significantly better than disease control groups at 3 months. However, at 6 months there was no significant difference amongst any of the groups in terms of mean articular scores.

Conclusion

Conclusion:Multiple injections are better than single injection at long term in terms of anti-inflammatory effect. multiple injections also exert chondroprotectice effect at short term