Siegmund Lang

The effect of leukocyte-reduced platelet-rich plasma on the proliferation of autologous adipose-tissue derived mesenchymal stem cells1

Clinical application of platelet-rich plasma (PRP) and stem cells has become more and more important in regenerative medicine during the last decade. However, differences in PRP preparations may contribute to variable PRP compositions with unpredictable effects on a cellular level. In the present study, we modified the centrifugation settings in order to provide a leukocyte-reduced PRP and evaluated the interactions between PRP and adipose-tissue derived mesenchymal stem cells (ASCs).PRP was obtained after modification of three different centrifugation settings and investigated by hemogram analysis, quantification of protein content and growth factor concentration. ASCs were cultured in serum-free α-MEM supplemented with autologous 10% or 20% leukocyte-reduced PRP. Cell cycle kinetics of ASCs were analyzed using flow cytometric analyses after 48 hours.Thrombocytes in PRP were concentrated, whereas erythrocytes, and white blood cells (WBC) were reduced, independent of centrifugation settings. Disabling the brake further reduced the number of WBCs. A higher percentage of cells in the S-phase in the presence of 20% PRP in comparison to 10% PRP and 20% fetal calf serum (FCS) advocates the proliferation stimulation of ASCs.These findings clearly demonstrate considerable differences between three PRP separation settings and assist in safeguarding the combination of leukocyte-reduced PRP and stem cells for regenerative therapies.

Leukocyte-Reduced Platelet-Rich Plasma Treatment of Basal Thumb Arthritis: A Pilot Study

A positive effect of intra-articular platelet-rich plasma (PRP) injection has been discussed for osteoarthritic joint conditions in the last years. The purpose of this study was to evaluate PRP injection into the trapeziometacarpal (TMC) joint. We report about ten patients with TMC joint osteoarthritis (OA) that were treated with 2 intra-articular PRP injections 4 weeks apart. PRP was produced using the Double Syringe System (Arthrex Inc., Naples, Florida, USA). A total volume of 1.47 ± 0.25 mL PRP was injected at the first injection and 1.5 ± 0.41 mL at the second injection, depending on the volume capacity of the joint. Patients were evaluated using VAS, strength measures, and the Mayo Wrist score and DASH score after 3 and 6 months. VAS significantly decreased from 6.2 ± 1.6 to 5.4 ± 2.2 at six-month follow-up (P < 0.05). The DASH score was unaffected; however, the Mayo Wrist score significantly improved from 46.5 ± 18.6 to 67.5 ± 19.0 at six-month follow-up (P = 0.05). Grip was unaffected, whereas pinch declined from 6.02 ± 2.99 to 3.96 ± 1.77 at six-month follow-up (P < 0.05). We did not observe adverse events after the injection of PRP, except one occurrence of a palmar wrist ganglion, which resolved without treatment. PRP injection for symptomatic TMC OA is a reasonable therapeutic option in early stages TMC OA and can be performed with little to no morbidity.

Autologous mesenchymal stem cells or meniscal cells: what is the best cell source for regenerative meniscus treatment in an early osteoarthritis situation?

BackgroundTreatment of meniscus tears within the avascular region represents a significant challenge, particularly in a situation of early osteoarthritis. Cell-based tissue engineering approaches have shown promising results. However, studies have not found a consensus on the appropriate autologous cell source in a clinical situation, specifically in a challenging degenerative environment. The present study sought to evaluate the appropriate cell source for autologous meniscal repair in a demanding setting of early osteoarthritis.MethodsA rabbit model was used to test autologous meniscal repair. Bone marrow and medial menisci were harvested 4 weeks prior to surgery. Bone marrow-derived mesenchymal stem cells (MSCs) and meniscal cells were isolated, expanded, and seeded onto collagen-hyaluronan scaffolds before implantation. A punch defect model was performed on the lateral meniscus and then a cell-seeded scaffold was press-fit into the defect. Following 6 or 12 weeks, gross joint morphology and OARSI grade were assessed, and menisci were harvested for macroscopic, histological, and immunohistochemical evaluation using a validated meniscus scoring system. In conjunction, human meniscal cells isolated from non-repairable bucket handle tears and human MSCs were expanded and, using the pellet culture model, assessed for their meniscus-like potential in a translational setting through collagen type I and II immunostaining, collagen type II enzyme-linked immunosorbent assay (ELISA), and gene expression analysis.ResultsAfter resections of the medial menisci, all knees showed early osteoarthritic changes (average OARSI grade 3.1). However, successful repair of meniscus punch defects was performed using either meniscal cells or MSCs. Gross joint assessment demonstrated donor site morbidity for meniscal cell treatment. Furthermore, human MSCs had significantly increased collagen type II gene expression and production compared to meniscal cells (p < 0.05).ConclusionsThe regenerative potential of the meniscus by an autologous cell-based tissue engineering approach was shown even in a challenging setting of early osteoarthritis. Autologous MSCs and meniscal cells were found to have improved meniscal healing in an animal model, thus demonstrating their feasibility in a clinical setting. However, donor site morbidity, reduced availability, and reduced chondrogenic differentiation of human meniscal cells from debris of meniscal tears favors autologous MSCs for clinical use for cell-based meniscus regeneration.

Leukocyte-reduced Platelet-rich Plasma Alters Protein Expression of Adipose Tissue–derived Mesenchymal Stem Cells

Background: Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue–derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. Methods: Adipose tissue–derived mesenchymal stem cells were cultured in &agr;-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum. Cell proliferation was assessed with cell cycle kinetics using flow cytometric analyses after 48 hours. Differences in proteome expression of the adipose tissue–derived mesenchymal stem cells were analyzed using a reverse-phase protein array to quantify 214 proteins. Complementary Ingenuity Pathways Analysis and gene set enrichment analysis were performed using protein data, and confirmed by Western blot analysis. Results: A higher percentage of adipose tissue–derived mesenchymal stem cells in the S phase in the presence of platelet-rich plasma advocates the proliferation stimulation. Ingenuity Pathways Analysis and gene set enrichment analysis confirm the involvement of the selected proteins in the process of cell growth and proliferation. Ingenuity Pathways Analysis revealed a participation in the top-ranked canonical pathways PI3K/AKT, PTEN, ILK, and IGF-1. Gene set enrichment analysis identified the authors’ protein set as being part of significantly regulated protein sets with the focus on cell cycle, metabolism, and the Kyoto Encyclopedia of Genes and Genomes transforming growth factor-&bgr; signaling pathway. Conclusions: The present study provides evidence that platelet-rich plasma stimulates proliferation and induces a unique change in the proteomic profile of adipose tissue–derived mesenchymal stem cells. The interpretation of altered expression of regulatory proteins represents a step forward toward achieving good manufacturing practice–compliant criteria for cell-based strategies.

Tissue Engineering of Large Full-Size Meniscus Defects by a Polyurethane Scaffold: Accelerated Regeneration by Mesenchymal Stromal Cells

The endogenous healing potential of avascular meniscal lesions is poor. Up to now, partial meniscectomy is still the treatment of choice for meniscal lesions within the avascular area. However, the large loss of meniscus substance predisposes the knee for osteoarthritic changes. Tissue engineering techniques for the replacement of such lesions could be a promising alternative treatment option. Thus, a polyurethane scaffold, which is already in clinical use, loaded with mesenchymal stromal cells, was analyzed for the repair of critical meniscus defects in the avascular zone. Large, approximately 7 mm broad meniscus lesions affecting both the avascular and vascular area of the lateral rabbit meniscus were treated with polyurethane scaffolds either loaded or unloaded with mesenchymal stromal cells. Menisci were harvested at 6 and 12 weeks after initial surgery. Both cell-free and cell-loaded approaches led to well-integrated and stable meniscus-like repair tissue. However, an accelerated healing was achieved by the application of mesenchymal stromal cells. Dense vascularization was detected throughout the repair tissue of both treatment groups. Overall, the polyurethane scaffold seems to promote the vessel ingrowth. The application of mesenchymal stromal cells has the potential to speed up the healing process.

Platelet-Rich Plasma in Tissue Engineering: Hype and Hope

Background: Platelet-rich plasma (PRP) refers to an enriched platelet suspension in plasma. In addition to the clinical application of PRP in the context of various orthopedic diseases and beyond, PRP and platelet lysate (PL) have been in focus in the field of tissue engineering. In this review, we discuss the application of PRP as a cell culture supplement and as part of tissue engineering strategies, particularly emphasizing current hurdles and ambiguities regarding the efficacy of PRP in these approaches. Summary: As a putative autologous replacement for animal-derived supplements such as fetal calf serum (FCS), PRP has been applied as cell culture supplement for the expansion of stem and progenitor cells for tissue engineering applications and cell therapies. Attributed to the high content of growth factors in platelets, PRP has been shown to promote cell growth, which was mostly superior to standard cultures supplemented with FCS, while the differentiation capacity of progenitor cells seems not to be affected. However, it was also suggested that cultivation of cells with PRP significantly alters the protein expression profile in cells in comparison to FCS, indicating that the influence of PRP on cell behavior should be thoroughly investigated. Moreover, different PRP preparation methods and donor variations have to be considered for the use of PRP under good manufacturing practice conditions. PRP has been used for various tissue engineering applications in the context of bone, cartilage, skin, and soft tissue repair, where most studies were conducted in the field of bone tissue engineering. These approaches take either advantage of the release of chemoattractive, angiogenic, proliferative, and putatively pro-regenerative growth factors from PRP, and/or the hydrogel properties of activated PRP, making it suitable as a cell delivery vehicle. In many of these studies, PRP is combined with biomaterials, cells, and in some cases recombinant growth factors. Although the experimental design often does not allow conclusions on the pro-regenerative effect of PRP itself, most publications report beneficial effects if PRP is added to the tissue-engineered construct. Furthermore, it was demonstrated that the release of growth factors from PRP may be tailored and controlled when PRP is combined with materials able to capture growth factors. Key Messages: Platelet-derived preparations such as PRP and PL represent a promising source of autologous growth factors, which may be applied as cell culture supplement or to promote regeneration in tissue-engineered constructs. Furthermore, activated PRP is a promising candidate as an autologous cell carrier. However, the studies investigating PRP in these contexts often show conflicting results, which most likely can be attributed to the lack of standardized preparation methods, particularly with regard to the platelet content and donor variation of PRP. Ultimately, the use of PRP has to be tailored for the individual application.

Partial Anterior Cruciate Ligament Ruptures: Advantages by Intraligament Autologous Conditioned Plasma Injection and Healing Response Technique—Midterm Outcome Evaluation

The historical treatment options for partial anterior cruciate ligament (ACL) ruptures were conservative therapy or ACL reconstruction by injured bundle or entire ACL replacement. In awareness of the regenerative potential of biologic agents such as mesenchymal stem cells or platelet rich plasma (PRP), the healing response technique was developed to preserve the injured ACL with better outcomes than the conservative therapy. Further improvement of this technique seems to be obtained by the additional application of PRP products. Thus, the aim of this study was to evaluate the midterm outcome after intraligament autologous conditioned plasma (ACP) by a clinical, scoring, and functional performance assessment. 42 patients were evaluated in this study. The failure rate was 9.5%. Outcome evaluation showed good to excellent results. The scores were IKDC subjective 83.2 (SD 14.5), Lysholm 85.5 (SD 15.5), Tegner 4.7 (SD 1.7), and Cincinnati 85.4 (SD 15.5) after a mean follow-up of 33 months. Clinical examination showed stable Lachman test, negative pivot shift phenomenon, and a significant reduction in AP-laxity compared to preoperative status (rolimeter preoperative: 1.9 (SD1.4); postoperative 0.6 (SD1.8), p=0.001) in all patients. Functional performance testing showed no significant differences between the injured and healthy side. Return to sport was achieved after a mean of 5.8 months (SD 3.6) in 71.1% of the included patients. In summary, this new treatment option revealed in midterm follow-up promising results to treat partial ACL lesions with a reduced need for conversion to ACL reconstruction and with a high percentage of return to preinjury sport activity.

Long-term radiographic appearance of calcium-phosphate synthetic bone grafts after surgical treatment of tibial plateau fractures

OBJECTIVES Synthetic bone grafts (SBGs) are widely used to fill bone defects after fracture reduction. This study assessed the long-term resorption of two different calcium phosphate products (A=ChronOS™ inject and B=Norian® SRS®; both DePuy Synthes, Oberdorf, Switzerland) used in the surgical treatment of tibial plateau fractures. DESIGN Long-term clinical and radiologic follow-up of 52 patients after surgical treatment of intraarticular tibial plateau fractures augmented with SBGs. SETTING The study was performed at a level 3 trauma center. PATIENTS Between January 2000 and December 2006 a total of 52 patients with intraarticular tibial plateau fractures were operatively treated and augmented with SBGs consisting of a Brushite matix with β-TCP granules (SBG A) or hydroxylapatite with 4-6% carbonate content (SBG B). 46 patients could be contacted and 38 were included in the study. Half of the patients received SBG A and the other half SBG B. MAIN OUTCOME MEASUREMENTS Loss of reduction and SBG resorption was investigated by comparison of follow-up X-ray images to pre- and postoperative X-ray images. Furthermore, pain, activity level and knee function were evaluated by means of questionnaires and clinical examination. RESULTS The mean age of patients was 59.7±12.5years. The follow-up was 8.6±0.9years for SBG A and 11.6±1.4years for SBG B (p<0.001). In most cases SBG A was completely resorbed in a homogenous pattern, while SBG B was still visible on the X-ray images revealing a peripheral resorption pattern. A loss of reduction (>2mm) could be observed in two patients with SBG A and two patients with SBG B, although only one of them had an impaired knee function. CONCLUSIONS The composite SBG A reveals a comprehensive long-term resorption in comparison to SBG B. Nevertheless, both provided suitable mechanical support as part of the surgical treatment of tibial plateau fractures. LEVEL OF EVIDENCE Case series, Level IV.

Leukocyte-reduced platelet-rich plasma stimulates the in vitro proliferation of adipose-tissue derived mesenchymal stem cells depending on PDGF signaling.

BACKGROUND Platelet-rich Plasma (PRP) is suggested as xenoprotein-free cell-culture medium replacement for animal-derived supplements. OBJECTIVE The aim of this study was to investigate PRP-triggered signaling in adipose derived mesenchymal stem cells (ASCs). METHODS PRP was obtained from 4 male patients. We incubated ASCs in α-MEM with different Platelet derived growth factor (PDGF) subtypes or 10% or 20% pooled PRP or 20% fetal calf serum (FCS) prior to determination of the S-phase fraction (SPF). To investigate the influence of PDGF signaling on ASCs, PDGF receptor β inhibitor was added, and protein expression of ASCs was measured. RESULTS ASCs exposed to 20% PRP, PDGF-AB and - BB demonstrated significant higher SPF in comparison to PDGF-AA and 20% FCS after 48 hours (all P < 0.05). PDGF receptor β inhibition diminished the PRP-induced SPF increase of ASCs significantly after 48 hours (P < 0.01). ASCs with PDGF receptor β inhibition showed significant higher PDGF receptor β and significant lower c-MYC expression compared to untreated cells in presence of 20% PRP after 48 hours (both P < 0.05). CONCLUSIONS The proliferation promoting effect of PRP on ASCs is mediated by PDGF signaling and is associated with c-MYC overexpression.