Sebastian Lippross

Intraarticular injection of platelet-rich plasma reduces inflammation in a pig model of rheumatoid arthritis of the knee joint.

OBJECTIVE Treatment options for rheumatoid arthritis range from symptomatic approaches to modern molecular interventions such as inhibition of inflammatory mediators. Inhibition of inflammation by platelet-rich plasma (PRP) has been proposed as a treatment for tendinitis and osteoarthritis. The present study was undertaken to investigate the effect of PRP on antigen-induced arthritis (AIA) of the knee joint in a large animal model. METHODS Six-month-old pigs (n = 10) were systemically immunized by bovine serum albumin (BSA) injection, and arthritis was induced by intraarticular BSA injection. PRP was injected into the knee joints of 5 of the animals after 2 weeks. An additional 5 animals received no systemic immunization (controls). Signs of arthritis were documented by plain histologic analysis, Safranin O staining, and immunohistochemistry analysis for type II collagen (CII), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF). Interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNFα), VEGF, and insulin-like growth factor 1 (IGF-1) protein content was measured by Luminex assay. RESULTS In the pigs with AIA, plain histologic analysis revealed severe arthritic changes in the synovium. Safranin O and CII staining showed decreased proteoglycan and CII content in cartilage. Immunohistochemistry analysis revealed increased levels of IL-6 and VEGF in synovium and cartilage, and protein concentrations of IL-6, VEGF, IL-1β, and IGF-1 in synovium and cartilage were elevated as well; in addition, TNFα protein was increased in cartilage. Treatment with PRP led to attenuation of these arthritic changes in the synovium and cartilage. CONCLUSION We have described a porcine model of AIA. Experiments using this model demonstrated that PRP can attenuate arthritic changes as assessed histologically and based on protein synthesis of typical inflammatory mediators in the synovial membrane and cartilage.

Platelets display potent antimicrobial activity and release human beta-defensin 2

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/109 platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.

Thrombocytes are effectors of the innate immune system releasing human beta defensin-3.

BACKGROUND Thrombocyte concentrate i.e. platelet-rich plasma (PRP) has become a popular adjunct for many surgical procedures. It is believed to improve bone and soft tissue healing. Recently antimicrobial effects of the autologous preparation were reported by several groups. In this study we investigated the antimicrobial effect of PRP against gram-negative microbes which frequently cause severe complications in orthopaedic trauma surgery. METHODS Platelet-rich plasma was produced from liquid preserved thrombocyte concentrates. ELISA, Western blot and immunohistochemistry were preformed to investigate the release and content of platelet concentrates. A radial diffusion assay was used to detect antimicrobial effects of PRP. RESULTS We detected the human beta defensin-3 in bactericidal concentrations in platelet preparations by ELISA, Western blot and immunohistochemistry. In antimicrobial testing we demonstrated effective inhibition of Escherichia coli (ATCC 11303), Bacterium megaterium (ATCC 14581), Klebsiella pneumoniae (ATCC 13883), Enterococcus faecalis (ATCC 29212) and Proteus mirabilis (ATCC21100). CONCLUSION With this study we demonstrate antimicrobial action of a popular adjunct for orthopaedic and trauma surgery against gram-positive and gram-negative bacteria. We have identified a possible mechanism of action via the secretion of HBD-3 as a first line defence in contaminated wounds and in elective application of PRP. This finding supports a broader spectrum of clinical indications for an autologous platelet preparation.

Sulforaphane has opposing effects on TNF-alpha stimulated and unstimulated synoviocytes

IntroductionRheumatoid arthritis (RA) is characterized by progressive inflammation associated with rampantly proliferating synoviocytes and joint destruction due to oxidative stress. Recently, we described nuclear factor erythroid 2-related factor 2 (Nrf2) as a major requirement for limiting cartilage destruction. NF-κB and AP-1 are the main transcription factors triggering the inflammatory progression in RA. We used sulforaphane, an isothiocyanate, which is both an Nrf2 inducer and a NF-κB and AP-1 inhibitor.MethodsCultured synoviocytes were stimulated with sulforaphane (SFN) with or without TNF-α pre-treatment. NF-κB, AP-1, and Nrf2 activation was investigated via dual luciferase reporter gene assays. Matrix metalloproteinases (MMPs) were measured via zymography and luminex technique. Cytokine levels were detected using ELISA. Cell viability, apoptosis and caspase activity were studied. Cell proliferation was analysed by real-time cell analysis.ResultsSFN treatment decreased inflammation and proliferation dose-dependently in TNF-α-stimulated synoviocytes. SFN did not reduce MMP-3 and MMP-9 activity or expression significantly. Interestingly, we demonstrated that SFN has opposing effects on naïve and TNF-α-stimulated synoviocytes. In naïve cells, SFN activated the cytoprotective transcription factor Nrf2. In marked contrast to this, SFN induced apoptosis in TNF-α-pre-stimulated synoviocytes.ConclusionsWe were able to show that SFN treatment acts contrary on naïve and inflammatory synoviocytes. SFN induces the cytoprotective transcription factor Nrf2 in naïve synoviocytes, whereas it induces apoptosis in inflamed synoviocytes. These findings indicate that the use of sulforaphane might be considered as an adjunctive therapeutic strategy to combat inflammation, pannus formation, and cartilage destruction in RA.

The Treatment of Intra-articular Calcaneus Fractures with Severe Soft Tissue Damage with a Hinged External Fixator or Internal Stabilization: Long-term Results

We developed a hinged external fixator for the treatment of dislocated intra-articular calcaneus fractures with severe soft tissue damage. The external fixation was performed with a known external fixator system. The screw insertion points were biomechanically tested by defining a virtual rotation axis through the center of the talus to allow early active motion in the ankle joint. Long-term follow-up was performed after an average of 7.3 years. Results were graded with the American Orthopaedic Foot and Ankle Society (AOFAS) score. Radiographs were reviewed according to Sanders classification. Four open fractures and 33 cases with extremely swollen soft tissue, blisters, or compartment syndromes were treated. In 24 cases (64.9%), the hinged fixator was the final method of treatment (group I). A change to open reduction with internal fixation was performed in 13 fractures (35.1%) when soft tissue problems were minimal (group II). There were no late amputations, osteomyelitis, or malunions. According to Sanders classification, group I consisted of 14 type II, 8 type III, and 2 type IV fractures. Pin loosening or pin infection was seen in 4 cases, but there was no redislocation. The Böhlers angle improved in 43%, gaps in the posterior facet were closed in 41%, and any shortening or deviation of the axis was corrected in 82% of the cases. The AOFAS score for the group averaged 66.5. According to Sanders classification, group II consisted of 8 type II and 5 type III fractures. The Böhlers angle improved in 88%, and gaps in the posterior facet were closed in 87%. Any shortening or deviation of the axis was corrected in 95%, and the AOFAS score averaged 61.3. Significant differences in patient outcome scores between open reduction with internal fixation and hinged fixator were not found. P value was > .05. The hinged external fixator frame can be used in all calcaneus fracture types without soft tissue limitation. The hinged fixator allows early movement in the ankle joint, the risk of infection is minimized, and secondary plate fixation remains possible.

The effect of platelet rich plasma on angiogenesis in ischemic flaps in VEGFR2-luc mice

To improve skin flap healing, one promising strategy in reconstructive surgery might be to optimize platelet rich plasma (PRP) bioactivity and the ischemia-altered expression of genes. We studied both the effect of PRP on ischemic flaps, and whether in vivo bioluminescence imaging (BLI) is a suitable method for the longitudinal monitoring of angiogenesis in surgical wounds. Axial murine skin flaps were created in four experimental groups. In vivo measurements of VEGFR2 expression levels were made every other day until the 14th day. The local VEGF level and microvessel density were quantified on the 14th day via ELISA and immunohistochemistry, and flap survival rates were measured. We demonstrated that PRP and induced ischemia have a beneficial influence on angiogenesis and flap healing. Combining the two resulted in a significantly robust increase in angiogenesis and flap survival rate that was corroborated by bioluminescence imaging of VEGFR2 activity. This study shows that angiogenic effects of PRP may be potentialized by the stimulus of induced ischemia during free flap harvesting, and thus the two procedures appear to have a synergistic effect on flap healing. This study further demonstrates that BLI of modulated genes in reconstructive surgery is a valuable model for longitudinal in vivo evaluation of angiogenesis.

Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes

Platelet‐released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet‐rich fibrin (PRF®)] are thrombocyte concentrate lysates that support healing of chronic, hard‐to‐heal and infected wounds. Human beta‐defensin‐2 (hBD‐2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound‐related bacteria. In this study, we analysed the influence of PRGF on hBD‐2 expression in human primary keratinocytes and the influence of Vivostat PRF® on hBD‐2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD‐2 gene and protein expressions in a concentration‐ and time‐dependent manner. The use of blocking antibodies revealed that the PRGF‐mediated hBD‐2 induction was partially mediated by the epidermal growth factor receptor and the interleukin‐6 receptor (IL‐6R). Luciferase gene reporter assays indicated that the hBD‐2 induction through PRGF required activation of the transcription factor activator protein 1 (AP‐1), but not of NF‐kappaB. In concordance with these cell culture data, Vivostat PRF® induced hBD‐2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD‐2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds.

Tuberculosis of the spine – Prospective neurological and patient reported outcome study

OBJECTIVE To prospectively investigate the rate of neurological recovery and patient reported outcome of tuberculosis (TB) spine patients following surgery at a tertiary referral hospital. TB spine remains a major cause of neurological impairment in the developing world fuelled by poor socio-economic conditions and HIV co-infection. Although numerous retrospective studies are available, there is a paucity of prospective data regarding rate of neurological improvement and patient reported outcome. METHODS Twenty adult patients were prospectively recruited. The average age was 44.5 years. Half had co-existent HIV infection. All patients underwent decompressive surgery utilising a variety of anterior and posterior procedures. All received a minimum of 9 months TB medication. They were followed up at 4, 8, 12, 26 and 52 weeks post operatively. Neurological status was monitored by Nurick, mJOA and ASIA systems. Patient reported outcome was monitored by SF36 questionnaires at all-time points. RESULTS All patients improved neurologically including 4 ASIA As. By last follow up, 17 could walk as opposed to 5 pre-op. The Nurick average score improved from 5.5 to 1.9 and the mJOA lower limb score 1.8-5.5. The SF36 improved from 31 to 62 over the year with the biggest gains occurring after 3 months. This was confirmed in all domains except pain and social, which improved earlier. CONCLUSIONS There is a positive prognosis for neurological outcome in TB spine following a variety of surgical decompressive procedures and medical therapy. The majority of the recovery occurs after the 3 months post-operative mark.

Reduction and retention of thoracolumbar fractures by minimally invasive stabilisation versus open posterior instrumentation.

PURPOSE The purpose of this study was in thoracolumbar fractures to assess the effectiveness of minimal invasive stabilisation compared to the open technique with regards to the change in kyphosis angle, the loss of reduction and length of hospital stay. METHODS The retrospective study consisted of 104 patients who received minimally invasive stabilisation or open stabilisation. Patients were between 15 and 86 years of age, had a thoracolumbar fracture and no neurological deficits. Kyphotic angle (Cobb angle) and loss of reduction was compared after minimal invasive and open stabilisation. The Cobb angle was evaluated directly post operatively, at 6 weeks, 3 months, 6 months and 12 months after surgery. RESULTS Evaluated patients who received the minimally invasive technique had a shorter surgical intervention time and a shorter hospital stay compared to patients who received the open technique. Kyphosis angle and loss of reduction showed no significant difference compared to open technique. There was also no significant difference between minimally invasive poly-axial and mono-axial stabilisation. CONCLUSION In this study we provide evidence that MIS instrumentation in selected thoracolumbar fractures can effectively be used without significant differences in loss of reduction compared to open stabilisation. MIS can also sufficiently retain reduction as compared to traditional open techniques. The main advantages are reduced operation time and shorter hospital stay.

Co-cultures of programmable cells of monocytic origin and mesenchymal stem cells do increase osteogenic differentiation.

Impaired bone healing can occur with numerous pathologic conditions like trauma, osteoporosis, and infection. Therefore tissue‐engineering strategies that aim to enhance osteogenic differentiation of stem cells in order to accelerate bone healing are a major goal of contemporary regenerative research. In this study we cultivated mesenchymal stem cells (MSC) together with the recently patented programmable cells of monocytic origin (PCMO) to test whether co‐cultures promote an osteogenic differentiation process. PCMO have recently been shown to have pluripotent characteristics and do support the regeneration processes of liver and heart diseases. Quantitative real time PCR expression profiles of osteogenic marker genes such as alkaline phosphatase in co‐cultures of PCMO and MSC showed that MSC differentiated into osteoblast‐like cells more rapidly as compared to mono‐cultures. Alkaline phosphatase expression and enzyme activity levels were highly increased in co‐cultures compared to mono‐cultures of MSC. Tests for mineralized matrix formation also indicated that PCMO have a positive effect on co‐cultured MSC under osteogenic culture conditions. However, analysis of collagen 1A did not show enhanced expression. In summary, PCMO obviously have the ability to promote osteogenic differentiation of MSC in vitro while their own pluripotent potential is not sufficient to develop osteoblast‐like characteristics themselves.