Effect of Human Serum and 2 Different Types of Platelet Concentrates on Human Meniscus Cell Migration, Proliferation, and Matrix Formation Undine Freymann, Ph.D., Sebastian Metzlaff, M.D., Jan-Philipp Krüger, Ph.D., Glen Hirsh, B.Sc., Michaela Endres, Ph.D., Wolf Petersen, M.D., and Christian Kaps, Ph.D.
Purpose: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. Methods: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. Results: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P ¼ .0001, 5% PRP: P ¼ .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P ¼ .0001) and 5% PRP (P ¼ .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P ¼ .0002, ACP: P ¼ .0147), cartilage oligomeric matrix protein (HS: P ¼ .0002, ACP: P ¼ .0005), and biglycan (HS: P ¼ .0002, ACP: P ¼ .0003), whereas PRP showed no inducing effect. Conclusions: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. Clinical Relevance: Our findings may suggest that HS might be a beneficial augment for meniscus repair.
M
enisci play a key role in the knee joint in terms of force transmission, shock absorption, provision of joint stability, and lubrication. Disruption and loss of the fibrous meniscus tissue increases the contact From the TransTissue Technologies GmbH, Department of Research & Development (U.F., J-P.K., G.H., M.E., C.K.), Berlin, Germany; Clinic for Traumatic Surgery and Orthopedics, Martin-Luther-Hospital (S.M., W.P.), Berlin, Germany; DeSimone Laboratory, Department of Cell Biology, University of Virginia (G.H.), Charlottesville, Virginia, U.S.A.; and Tissue Engineering Laboratory, Department of Rheumatology and Immunology, Charité - University Hospital Berlin (M.E.), Berlin, Germany. The authors report the following potential conflict of interest or source of funding: U.F., J-P.K., and M.E. receive support from TransTissue Technologies GmbH. W.P. receives support from Clinical Excellence Circle, Otto Bock Health Care, Karl Storz Endoscopy, and AAP Implants. C.K. receives support from TransTissue Technologies GmbH and BioTissue AG. Received January 30, 2015; accepted November 17, 2015. Address correspondence to Undine Freymann, Ph.D., TransTissue Technologies GmbH, Charitéplatz 1, 10117 Berlin, Germany. E-mail: undine. [email protected] Ó 2016 by the Arthroscopy Association of North America 0749-8063/1591/$36.00 http://dx.doi.org/10.1016/j.arthro.2015.11.033
pressure on the articular cartilage and can cause longterm articular cartilage injuries followed by dysfunction and secondary osteoarthritis.1 Annually, more than 1.5 million meniscus procedures are performed across the United States and Europe that most often include partial meniscectomy for irreparable meniscus lesions.2 In recent years, there has been a growing interest in using regenerative medicine approaches to repair damaged meniscus tissue.3 Thereby, optimal healing may rely on (1) a mechanical scaffold for cell adherence and for supporting new tissue formation, (2) the appropriate cell type to initiate tissue healing and new matrix synthesis, and (3) signals to stimulate cell differentiation and expression of matrix genes.4,5 Bloodderived products are considered as an attractive autologous source of such stimulatory signals. Consequently, blood-derived products such as human serum (HS)6 and different types of platelet concentrates7,8 have been used in vitro and in vivo to support mesenchymal tissue regeneration.
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The migration and differentiation of cells may be mediated by cytokines and growth factors. Recent in vitro studies showed a recruiting and differentiating effect of HS on human meniscus cells and meniscus-like matrix formation in high-density cultures as well as in 3-dimensional polyglycolic acid/hyaluronan scaffolds.9,10 Platelet-rich plasma (PRP) preparations are defined as suspensions of platelets in plasma, characterized by a platelet concentration that is higher than that of the original blood.11 In brief, platelets, once activated, release growth factors from their alphagranules that play an important role in the healing process and in the formation of different tissue types.7 Thus, they are also an attractive tool for the treatment of meniscal lesions. Different types of platelet concentrates can be prepared either by centrifugation methods or by commercially available separation kits (e.g., Autologous Conditioned Plasma [ACP]), which are designed for direct intraoperative use. ACP is prepared by a separation kit and described as a low-concentrate leukocyte-poor platelet preparation in contrast to those prepared by standard centrifugation methods that are characterized by a high amount of platelets and a low leukocyte content (called PRP in this study).11 Aiming at a cell-free scaffold-based approach for clinical meniscus repair, we follow the route that such a repair attempt may be improved by cell recruitment followed by cell growth and meniscal extracellular matrix (ECM) formation. Therefore, the purpose of this study was to evaluate the effect of 10% HS, 5% PRP, and 5% ACP on migration, proliferation, and ECM synthesis of human meniscus cells. We hypothesized that, because of the different growth factor content of HS, PRP, and ACP, these 3 blood-derived products might show different stimulatory potentials for cell migration, proliferation, and ECM formation of human meniscus cells.
Methods Preparation of Human Meniscus Cells, Human Serum, and Platelet Concentrates Patients (male or female; >18 years; n ¼ 5; Department of Orthopedics, Martin-Luther Hospital Berlin, Germany) scheduled for total knee arthroplasty (TKA) or postmortem donors (male or female; >18 years; n ¼ 5; Department of Pathology, Charité - University Hospital Berlin, Germany) were included. From each patient/donor, 1 lateral meniscus with macroscopic signs of degeneration such as tissue defibration and/or fatty degeneration was used. Menisci from postmortem donors were resected within 24 hours after death. Mensici were transferred immediately to the laboratory in Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) containing 100 U/ml penicillin and 100 mg/ml streptomycin (1% pen/strep;
Biochrom) to perform cell isolation. Human meniscus cells were isolated by enzymatic digestion in DMEM containing 1% pen/strep, 1% gentamycin, 0.04% amphotericin B (all Biochrom), 10% HS (DRK Blutspendedienst, Berlin, Germany), 10,000 U collagenase II (Biochrom), and 45 U collagenase P (Roche, Basel, Switzerland) for 18 to 20 hours in an incubator (37 C, 95% humidity, and 5% CO2). Cells were cryopreserved in passage 0. To obtain the required cell number (46 106 cells for pellet cultures, 12 106 cells for proliferation assay) and to ensure comparable experimental conditions (same cell pool), cells had to be expanded. For preparation of the cell pools, cells were thawed, seeded at a cell density of 10,000 cells/cm2, and cultured up to passage 3 in DMEM containing 1% pen/ strep and 10% HS. The study was approved by the ethical review board of the Charité - University Hospital Berlin. HS was prepared from 450 ml whole blood samples without anticoagulant of 10 healthy, anonymous blood donors aged between 18 and 65 years (DRK Blutspendedienst). After coagulation, the serum fraction (approximately 200 ml) was separated from the blood clot and residual blood cells and fibrinogen were removed by additional centrifugation (4,400 rpm for 10 minutes). Then, HS was heat-inactivated for 30 minutes at 56 C in a water bath. Ten individual HS preparations were pooled for further experiments. PRP (high-concentrate leukocyte-poor platelet preparation) is a licensed drug (Thrombocyte Concentrate N-W, DRK Blutspendedienst), which was prepared by centrifugation methods. First initial centrifugation of whole blood from healthy, anonymous blood donors aged between 18 and 65 years was performed to remove cellular elements from the plasma. Thereby, 90% to 95% of platelets were concentrated along with the leukocytes in the buffy coat layer above the tightly packed erythrocyte infranatant. During further processing, the platelet-enriched buffy coat was separated and resuspended in a platelet additive solution. Four buffy coats were pooled on the day after collection and centrifuged to remove contaminating leukocytes and erythrocytes. Pooled buffy coat-derived platelet concentrates were passed through a leukoreduction filter to reduce the residual leukocytes to less than 1 106 cells. The final platelet preparation (PRP) contained functional intact and high-concentrated platelets (3.2 1011 platelets) as an active agent as specified by the manufacturer. For our studies, 3 of these PRP preparations were pooled in equal amounts before use. ACP (low-concentrate leukocyte-poor platelet preparation) was prepared by a commercially available separation kit (ACP Double Syringe System, Arthrex, Munich, Germany) from 3 healthy blood donors (age 27 to 35 years, 2 males, 1 female). Per donor, a 15 ml venous blood sample was collected slowly in a special
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
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double syringe provided by the manufacturer. Blood was mixed in the syringe with 1 ml of anticoagulant citrate dextrose-A (Sigma-Aldrich, Darmstadt, Germany) to prevent anticoagulation. The double syringe was placed in 1 bucket of a customized centrifuge and a single centrifugation was performed (1,500 rpm for 5 minutes). At the end, the supernatant ACP (3 to 4 ml) was transferred from the larger outer syringe into the small inner syringe, carefully avoiding mixing. The preparation procedure was performed 3 times per donor to increase the final ACP volume per donor. For further experiments, ACP preparations from 3 individual blood donors were pooled in equal amounts before use. All pools of blood-derived products (HS, PRP, ACP) were stored at 20 C and thawed before use. PRP and ACP pools were thawed slowly at 4 C, followed by centrifugation at 4 C at 1,600 g for 10 minutes to remove residual fibrinogen. Supernatants were used immediately for further experiments.
as control media. After 24 hours, the medium was replaced by DMEM containing ITSþ1 supplemented with either 10% HS, 5% PRP, or 5% ACP (n ¼ 3 per time point and experimental group) and cells were maintained for up to 9 days. Concentrations of tested blood-derived products were chosen on the basis of the results from chemotaxis assays and previous studies.9 For DNA analysis, harvested cells were incubated with 1 ml papain-cystein hydrochloride (0.125 mg/ml in distilled water) for 16 hours at 60 C. The supernatant was stored at 20 C. Samples were diluted (1:2 to 1:8, v/v) in 2 M sodium chloride and 0.05 mM sodium hydrogen phosphate (all Sigma-Aldrich). Serial dilutions of calf thymus DNA (Life Technologies, Darmstadt, Germany) were used to prepare a standard curve and to calculate the DNA content of samples. One hundred microliters of standard or sample was mixed with 100 ml of 0.67 mg/ml bisbenzimide (Life Technologies) and measured at 360 nm with light emission at 460 nm.
Cell Migration Assays The chemotactic effect of HS, PRP, and ACP on human meniscus cells (cell pool of 5 postmortem donors, 3 104 cells/well) was analyzed using an 8-mm-pore polycarbonate membrane assay (96-well ChemoTx, Neuro Probe, Gaithersburg, MD) according to the manufacturer’s recommendations. Concentrations between 1% and 10% of PRP and ACP were analyzed each in biological triplicates for 3 individual blood donors/preparations. Migratory activity of cells toward HS was tested only for 10% HS. The migratory effect of rising concentrations of HS (0.1% to 100%) has been reported previously and showed that 10% HS recruited the most human meniscus cells.9 Different concentrations of blood-derived products were prepared by dilution in DMEM (low glucose) containing 0.1% HS. This medium also served as control. After 20 hours at 37 C, cells that attached underneath the polycarbonate membrane were fixed with acetone/methanol 1:1 (v:v) for 3 minutes and counterstained with Hemacolor Rapid Stain (Merck, Darmstadt, Germany). Cells that migrated through the filter pores were analyzed microscopically. Pictures were taken from 3 representative visual fields of each well, stained cells were counted using ImageJ (National Institute of Health, Bethesda, MD), and the cell number was extrapolated to the size of the well.
Studies on Meniscal Extracellular Matrix Formation The influence of HS, PRP, and ACP on ECM formation was evaluated in a standard high-density pellet assay (cell pool of 5 TKA patients, 250,000 cells/pellet), as described previously.9 ECM formation was induced by adding either 10% HS, 5% PRP, or 5% ACP to DMEM (high glucose) containing 1% ITSþ1, 1 mM sodium pyruvate, 0.35 mM L-proline, 0.17 mM Lascorbic acid-2-phosphate, and 0.1 mM dexamethasone (all Sigma-Aldrich), 1% pen/strep and 2% 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (Biochrom). Pellets cultured in complete DMEM without PRP, ACP, or HS served as controls. Fifty-two pellets were prepared for each group (PRP, ACP, HS, control). The medium was exchanged every 2 to 3 days and cells were maintained for up to 28 days. For histological evaluation of ECM deposition, pellets were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA), and frozen and cryo-slides (6 mm) were prepared. Avascular tissue samples of human meniscus, which have been prepared from the original degenerative donor tissue before cell isolation, served as positive control. H&E staining was performed to assess pellet and cell morphology. Proteoglycans were stained with Alcian Blue 8GX (Carl Roth, Karlsruhe, Germany), followed by counterstaining with nuclear fast red (SigmaAldrich). For collagen type I and type II stainings, cryoslides were incubated for 40 minutes with mouse antihuman type I or rabbit antihuman type II collagen antibodies (Acris, Herford, Germany). Mouse and rabbit immunoglobulin G served as control (DAKO, Hamburg, Germany). Detection was performed using the EnVisionþþ System HRP Kit (DAKO), followed by counterstaining with hematoxylin. For each staining,
Cell Proliferation Studies The effect of HS, PRP, and ACP on cell proliferation was evaluated using a pool of meniscal cells (cell pool of 5 TKA patients). Cells were seeded in T25 cell culture flasks (10,000 cells/cm2) in DMEM (low glucose) containing 1% insulin-transferrin-sodium selenite with linoleic acid (ITSþ1; Sigma Aldrich), which also served
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n ¼ 3 pellets per group and n ¼ 3 slides per pellet were analyzed. Histomorphometric analysis was performed by 1 observer using the Adobe Photoshop Software as described.12 In brief, a standard color was defined that represents the particular color of the specific staining. The amount of stained pixels in relation to the total amount of pixels of the section represents the percentage of the positively stained area. Gene Expression Analysis Expression levels of collagen type I and II, aggrecan, biglycan, cartilage oligomeric protein (COMP), and biglycan, which have been shown to be expressed in native meniscus tissue,10 were examined in pellets at early day 14 (pool of n ¼ 20 pellets per experimental group). Total RNA was isolated and 590 ng was transcribed into single strand cDNA using the iScript Kit (BIO-RAD Laboratories, Munich, Germany) according to the manufacturer’s recommendations. Real-time reverse transcriptase polymerase chain reaction was performed using the “SYBR Green PCR Core Kit” (Life Technologies) and a PCR cycler (iCycler, BIO-RAD). The relative expression of glyceraldehyde-3-phosphate dehydrogenase was used to normalize the cDNA samples. The fold change (FC) of expression values for a given marker gene per group was calculated in relation to controls.
Table 1. Patient and Donor Characteristics Cell Pool (n ¼ 5) 1
2
66 68 77 78 79 48 85 56 60 69
Age, yr mean 73.6
mean 63.6
Sex (Male/ Female) f f f m m f f m m m
f, female; m, male.
Statistical Analysis Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH, Erkrath, Germany). The Kolmogorov-Smirnov method was used to determine normal distribution and equal variance of data. Subsequently, 1-way analysis of variance and multiple comparison procedure according to the StudentNewman-Keuls method were used for statistical analysis of data within 1 group as well as for intergroup analysis. Significant differences were considered at P < .05. For gene expression analysis, differential expression was considered at an FC of less than 2 or greater than 2 and P < .05. Bars present the mean value of data with standard deviation.
Results Patient and Donor Characteristics Patient and donor characteristics are given in Table 1. Chemotactic Effect of HS, PRP, and ACP on Human Meniscus Cells All blood-derived preparations showed a migratory effect on human meniscus cells (Fig 1A). Stimulation with 10% HS significantly recruited meniscus cells, 13,308 25 cells (mean standard deviation) compared with nonstimulated controls (5,388 1,254 spontaneously migrating cells). Stimulation with 1% PRP (10,142 2,755 cells) to 10% PRP (17,861
Fig 1. Migratory effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on human meniscus cells. (A) Migratory effects have been quantified by enumerating the number of migrating cells on stimulation with HS, ACP, and PRP. All 3 blood-derived products increase, to different extents, the number of migrating human meniscus cells. (B) Intergroup comparison between 10% HS, 5% PRP, 5% ACP, and nonstimulated controls. Asterisks (*) indicate significant differences (P > .05). The bars present the mean value of data with standard deviation (pool of n ¼ 5 donors) of each group.
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
2,312 cells) showed significantly increased cell numbers when compared with controls and significant differences in migrating cells between PRP concentrations of 1% to 10%. Migratory activity significantly increased on stimulation with 1% ACP (11,821 1,305 cells) to 10% ACP (11,417 2,963 cells) when compared with controls, but did not show a dose-dependent increase in the number of migrating cells. Further intergroup comparison (Fig 1B) showed significantly increased cell numbers after stimulation with 5% PRP, 5% ACP, and 10% HS in comparison with nonstimulated controls. Therein, stimulation with 5% PRP led to the highest migratory activity of cells and showed significantly increased cell numbers in comparison with the 10% HS and 5% ACP group as well. Proliferative Effect of HS, PRP, and ACP on Human Meniscus Cells Cell proliferation was evident after stimulation of human meniscus cells with 10% HS, 5% PRP, and 5% ACP as well as in nonstimulated controls as shown by the increase of DNA content (Fig 2A). The DNA content significantly (P < .05) increased over cultivation time (day 0 to 9) within the HS group (1.60 0.06 to 6.08 0.12 mg/ml) and PRP group (1.56 0.23 to 5.91 0.12 mg/ml). Stimulation with 5% ACP significantly increased DNA values at day 6 (2.71 0.35 mg/ml) and day 9 (3.04 0.12 mg/ml) when compared with day 0. In nonstimulated controls, the DNA content significantly increased at day 6 (2.42 0.99 mg/ml) and at day 9 (3.04 0.35 mg/ml) when compared with the values at day 0. At day 9 (Fig 2B), the DNA content significantly increased in cultures stimulated with HS (6.08 0.12 mg/ml) and PRP (5.91 0.12 mg/ml) when compared with the ACP group (3.04 0.12 mg/ml) or with the nonstimulated control group (3.04 0.35 mg/ ml). In the ACP group, no proliferative stimulus could be detected after 9 days. Effects of HS, PRP, and ACP on Extracellular Matrix Formation All 4 groups of human meniscus pellet cultures (10% HS, 5% ACP, 5% PRP, control) showed ECM formation at day 28 (Fig 3). The morphology and dimensions of the pellets are presented in Figure 3A. Therein, nonstimulated controls showed a whiter and more oval appearance of the pellets compared with the other groups. Stimulation with PRP or ACP resulted in an enlargement of pellets compared with the round compact form of HS-stimulated pellets. Largest, but also most inhomogeneous, pellets were found on stimulation of cell pellets with ACP, followed by the PRP group. Evaluation of ECM formation by H&E staining (Fig 3B) showed an ECM rich in cells in nonstimulated controls. Pellets stimulated with HS developed a well-structured
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ECM, rich in homogeneously distributed cells. PRPstimulated pellets formed an ECM with a cell-rich and compact part (Fig 3B, double black arrowhead) but also unstructured parts, poor in cells (Fig 3B, asterisk). ACPstimulated pellet cultures formed an ECM with a small and compact core rich in cells (Fig 3B, black arrowhead), surrounded by high amounts of unstructured ECM poor in cells (Fig 3B, asterisk). Histological staining of ECM components at day 28 (Fig 4) revealed that high-density pellets stimulated with HS developed a meniscus-like ECM with deposition of collagen type I, proteoglycans, and collagen type II (Fig 4 D-F) similar to findings in native meniscus tissue sections of the same donors (Fig 4 M-O). Pellets treated with PRP (Fig 4 G-I) or ACP (Fig 4 J-L) showed the formation of a fibrous and inhomogeneous ECM with weak deposition of collagen type I, proteoglycans, and collagen type II, located especially in the cell-rich ECM area and not in the surrounding fibrous parts. Nonstimulated controls (Fig 4 A-C) also showed the deposition of collagen type I and faint stainings of proteoglycans and collagen type II. Histomorphometric quantification at day 28 (Table 2) showed significant differences in collagen type I stained areas between nonstimulated controls and all other 3 stimulated groups as well as a significant decreased deposition of collagen type I in ACP-stimulated pellets in comparison with the HS and PRP groups. Histomorphometric quantification of proteoglycans (alcian blue staining) showed a significant increase in HSstimulated pellets when compared with controls, with the ACP group, and with the PRP group. ACP- and PRPstimulated pellets showed significant less proteoglycans than nonstimulated controls. The HS group showed the highest and the ACP group the weakest deposition of proteoglycans among all groups. Quantification of collagen type II stained areas showed a significant increase in HS-stimulated pellets when compared with the ACP and PRP groups. ACP- and PRP-stimulated pellets showed significant less collagen type II deposition than nonstimulated controls. Among all groups, HS-stimulated pellets showed the highest and ACPstimulated pellets the weakest deposition of collagen type II. Gene Expression Analysis of Human Meniscus Pellet Cultures To assess the effects of HS, PRP, and ACP on gene expression level, analysis of meniscus-related genes was performed at early day 14 (Fig 5). Stimulation of pellets with HS significantly induced the expression of aggrecan (FC ¼ 8.9 1.13), COMP (FC ¼ 6.07 0.73), and biglycan (FC ¼ 2.71 0.26) at day 14 compared with controls and also led to significantly elevated aggrecan and COMP expression levels in comparison with ACPand PRP-stimulated pellets. Stimulation of pellets with
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Fig 2. Proliferative effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on human meniscus cells. (A) Proliferative effects have been evaluated by measuring the content of genomic DNA of cells on stimulation with HS, PRP, and ACP for up to 9 days. DNA content and thus the number of progenitor cells increased after stimulation with HS and PRP. Nonstimulated controls and cells stimulated with ACP showed a comparable DNA content. (B) Intergroup comparison between all groups at day 9. Asterisks (*) indicate significant differences (P > .05). The bars present the mean value of data with standard deviation (pool of n ¼ 5 donors) of each group.
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Fig 3. Effect of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on meniscal extracellular matrix (ECM) formation at day 28. (A) Morphology and dimension of human meniscus pellet cultures stimulated with HS, PRP, or ACP, and nonstimulated controls. (B) H&E staining. Stimulation with HS showed formation of compact pellets with homogeneous and cell-rich ECM, whereas stimulation with ACP resulted in a large inhomogeneous pellet with a small area of cell-rich ECM (black arrowhead) surrounded by an unstructured, fibrous, and cell-poor ECM in the outer zone. PRP-stimulated pellet cultures showed a compact ECM area rich in cells (double black arrowhead), surrounded by unstructured fibrous ECM parts (asterisks).
ACP significantly induced the expression of aggrecan (FC ¼ 2.87 0.39), COMP (FC ¼ 3.49 0.35), and biglycan (FC ¼ 2.12 0.15) at day 14 compared with controls, but to a lesser extent than HS. PRP showed no effect on gene expression levels of aggrecan, COMP, or biglycan and led to a significantly decreased expression of collagen type I (FC ¼ 0.37 0.05) at day 14 compared with controls. Collagen type II (data not shown) and collagen type I expression levels were not induced by stimulation of pellets with HS, PRP, or ACP at early day 14 compared with nonstimulated controls.
Discussion In the current study, blood-derived products such as HS, PRP, and ACP enhanced cell migration, whereas cell proliferation was significantly increased only by HS and PRP. Gene expression analyses of pellet cultures at day 14 revealed an induction of genes coding for large and small meniscal proteoglycans like aggrecan and biglycan and other ECM proteins like COMP after stimulation with HS and ACP, although to a lesser extent. PRP did not show an inducing effect on fibrochondrogenic marker gene expression. An induction of collagen type I and type II expression was not detectable at early day 14 in any stimulated group. Despite that, there is evidence that gene transcription and protein translation occurred to a later time point, because we histologically evaluated the deposition of collagen type I and type II at
day 28. Formation of a well-structured meniscus-like ECM was stimulated only by HS, whereas PRP and ACP led to the formation of an unstructured and more fibrous ECM. The proliferative effect of PRP and HS on human meniscus cells is in line with other in vitro studies, showing a positive mitogenic effect on monolayer rabbit meniscal cells,13 sheep meniscal cell cultures,2 or human meniscus cells.10,14 Besides the impact on cell proliferation, all 3 bloodderived products have been shown to significantly stimulate the migration of human meniscus cells. The chemotactic effect of platelet concentrates is comparable with its effect on human subchondral mesenchymal progenitor cells15 or human tenocytes.16 The chemotactic effect of HS on human meniscus cells has been reported previously, where doses between 1% and 20% HS effectively attracted human meniscus cells,9 and is confirmed again by this study for 10% HS. Our results are comparable with other studies, which showed the effects of fetal bovine serum on cell migration and proliferation of porcine meniscus cells in micro-wound and repair models.17 The effect of HS may be due to its protein composition including cytokines, growth factors, and/or diverse chemokines, which have been shown to stimulate chemotaxis of cells.18 Also various growth factors have been proven to be effective in meniscus in vitro studies (Table 3).
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Fig 4. Histological and immunohistochemical evaluation of meniscal extracellular matrix (ECM) formation in human meniscus pellet cultures at day 28. Effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on ECM formation have been evaluated by histological and immunohistochemical analyses. Alcian blue and collagen type I and type II staining showed that nonstimulated controls showed high deposition of (A) collagen type I and faint stainings for (B) proteoglycans and (C) collagen type II. Cells stimulated with HS formed a meniscus-like ECM with high deposition of (D) collagen type I, formation of (E) proteoglycans, and high deposition of (F) collagen type II. (G-L) Cells stimulated with PRP or ACP showed inhomogeneous ECM formation. Therein, deposition of (H, K) proteoglycans and collagen (G, J) type I and (I, L) type II was found only in nonfibrous ECM areas. (M-O) Stained sections of the inner part of the original degenerative meniscus tissue samples served as positive control. Tissue samples showed homogenously distributed cells and the presence of (N) proteoglycans and collagen (M) type I and (O) type II.
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MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
Table 2. Analysis of Histomorphometric Data (Day 28) Using 1-Way Analysis of Variance and Multiple Comparison Procedure According to the Student-Newman-Keuls Method Comparison Group 1/Group 2 Collagen type I stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control Alcian blue stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control Collagen type II stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control
Mean SD (Group 1)/Mean SD (Group 2)
P Value
Significance (P < .05)
57.0 57.0 57.0 42.7 42.7 19.6
21.6/76.9 21.6/42.7 21.6/19.6 19.2/76.9 19.2/19.6 11.9/76.9
4.1 19.2 11.9 4.1 11.9 4.1
.0170 .0721 .0002 .0004 .0043 .0002
Yes No Yes Yes Yes Yes
57.4 57.4 57.4 26.1 26.1 6.1
10.4/43.3 11.0 10.4/26.1 9.9 10.4/6.1 5.9 9.9/43.3 11.0 9.9/6.1 5.9 5.9/43.3 11.0
.0049 .0001 .0002 .0018 .0005 .0001
Yes Yes Yes Yes Yes Yes
79.7 79.7 79.7 30.2 30.2 16.8
18.7/67.5 31.2 18.7/30.2 13.5 18.7/16.8 6.4 13.5/67.5 31.2 13.5/16.8 6.4 6.4/67.5 31.2
.2419 .0002 .0002 .0001 .1737 .0002
No Yes Yes Yes No Yes
ACP, autologous conditioned plasma; HS, human serum; PRP, platelet-rich plasma; SD, standard deviation.
The effects of HS, PRP, and ACP on gene expression and meniscus-like ECM formation are differing. Stimulation of meniscus cells with HS induced meniscus-related genes (aggrecan, biglycan, COMP) and led to the formation of a homogeneous meniscal ECM. Results are in line with previous studies, which showed a fibrocartilaginous gene expression profile and meniscus-like ECM formation after stimulation of human meniscus cells with HS in pellet and scaffold cultures.9,10 Small and large proteoglycans like biglycan and aggrecan have been stated to resist compressive and tensile forces in the adult human meniscus.10 Thus, the use of HS with its proven stimulatory potential to induce expression and deposition of meniscal ECM in vitro might also improve meniscus repair approaches in vivo. Platelet concentrates like PRP and ACP could not lead to comparable results on protein or gene expression level. Stimulation with ACP led to an early gene induction of aggrecan, biglycan, and COMP, but finally only resulted in the formation of an inhomogeneous and nonmeniscal ECM after 28 days. Stimulation with PRP led to the formation of a better structured, but still more fibrous ECM and showed no induction of fibrochondrogenic genes. Gene expression results for platelet preparations are comparable with those reported by others, where mRNA expressions have been measured after monolayer cultivation of rabbit meniscus cells with 10% PRP.13 However, in the same report, a gelatin gel immersed with PRP effectively repaired full-thickness meniscal defects in the rabbit model.13 In contrast to that, no significant
improvement was found when platelet preparations were used with a hyaluronan-collagen composite matrix for the treatment of rabbit meniscal defects.20 Obviously, further studies are needed to elucidate whether platelet preparations like PRP or ACP are beneficial for meniscus regeneration. In summary, HS and platelet concentrates like PRP and ACP as a source of growth factors showed different potentials to stimulate migration, proliferation as well as meniscus-like ECM formation of human meniscus cell in vitro. Limitations The current study has some limitations. This in vitro study does not reflect the in vivo situation. Therefore, our in vitro study cannot predict the effects or activities of blood-derived products like HS, PRP, or ACP in vivo. All studies were performed using a pool of 5 individually cell preparations and with pools of blood-derived products. Because of limited donor tissue availability, we used degenerative whole meniscus tissue derived from old donors with age-related degenerative changes, which do not represent the typical younger patient cohort for meniscus repair or replacement. We also did not discriminate between degenerative status and distinct meniscus regions. On the basis of the results of our migration experiments, we tested only a singular concentration for studies on proliferation and ECM formation and did not evaluate differential effects of different doses. Our study is also limited by only 1 reviewer for evaluation of histological findings and by a low sample size in some groups and varying blood
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Fig 5. Real-time gene expression analysis of meniscus marker genes at day 14. Fold change analysis of meniscal marker genes like collagen type I, aggrecan, cartilage oligomeric protein (COMP), biglycan, and collagen type II expressed in human meniscus pellet cultures compared with nonstimulated controls. Asterisks represent significant (P < .05) differences in gene expression between all 4 groups (human serum [HS], platelet-rich plasma [PRP] or autologous conditioned plasma [ACP], and nonstimulated controls). The bars show the mean fold change (n ¼ 3; pool of n ¼ 20 pellets per experimental group) of each group and standard deviation is plotted.
donors among the groups. The study would have been strengthened by using preparations derived from blood drawn of the same donors to probably elicit different biological activities of the different blood-derived products.
Conclusions Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of
Table 3. Growth Factor Application in Meniscus In Vitro Studies Growth Factor
Abbreviation
Study Outcome: In Vitro Effect of Growth Factor
Reference
Platelet-derived growth factor Transforming growth factor-beta 1 Fibroblast growth factor
PDGF TGF-ß1 FGF
Moran et al.2 (review)
Insulin-like growth factor
IGF
Vascular endothelial growth factor
VEGF
Ovine meniscus 3D scaffold cultures: stimulation of cell proliferation and migration, increase in collagen type I expression, decrease in collagen type II production Ovine meniscus 3D scaffold cultures: stimulation of cell proliferation, increase in collagen type I expression Ovine/bovine meniscal explant model: increase in DNA and extracellular matrix synthesis and induction of cell migration Ovine meniscus cells from the avascular zone: stimulation of collagen type I and type II synthesis
3D, 3-dimensional.
Moran et al.2 (review)
Esparza et al.19 2012
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches.
10.
Acknowledgment The authors thank Samuel Vetterlein and AnneCathrin Behr for excellent technical and laboratory assistance. This study was supported by the Bundesministerium für Wirtschaft und Technologie, project “Zentrales Innovationsprogramm Mittelstand,” grant ID: KF2699502FR2.
References 1. Walker PS, Arno S, Bell C, Salvadore G, Borukhov I, Oh C. Function of the medial meniscus in force transmission and stability. J Biomech 2015;48:1383-1388. 2. Moran CJ, Busilacchi A, Lee CA, Athanasiou KA, Verdonk PC. Biological augmentation and tissue engineering approaches in meniscus surgery. Arthroscopy 2015;31:944-955. 3. Liu CX, Toma IC, Mastrogiacomo M, Krettek C, von Lewinski G, Jagodzinski M. Meniscus reconstruction: Today’s achievements and premises for the future. Arch Orthop Trauma Surg 2013;133:95-109. 4. Taylor SA, Rodeo SA. Augmentation techniques for isolated meniscal tears. Curr Rev Musculoskelet Med 2013;6:95-101. 5. Makris EA, Hadidi P, Athanasiou KA. The knee meniscus: Structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials 2011;32:7411-7431. 6. Dhollander AA, Verdonk PC, Lambrecht S, et al. The combination of microfracture and a cell-free polymerbased implant immersed with autologous serum for cartilage defect coverage. Knee Surg Sports Traumatol Arthrosc 2012;20:1773-1780. 7. Dhillon MS, Behera P, Patel S, Shetty V. Orthobiologics and platelet rich plasma. Indian J Orthop 2014;48:1-9. 8. Pifer MA, Maerz T, Baker KC, Anderson K. Matrix metalloproteinase content and activity in low-platelet, lowleukocyte and high-platelet, high-leukocyte platelet rich plasma (PRP) and the biologic response to PRP by human ligament fibroblasts. Am J Sports Med 2014;42:1211-1218. 9. Freymann U, Endres M, Goldmann U, Sittinger M, Kaps C. Toward scaffold-based meniscus repair: Effect of
11.
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human serum, hyaluronic acid and TGF-ss3 on cell recruitment and re-differentiation. Osteoarthritis Cartilage 2013;21:773-781. Freymann U, Endres M, Neumann K, Scholman HJ, Morawietz L, Kaps C. Expanded human meniscus-derived cells in 3-D polymer-hyaluronan scaffolds for meniscus repair. Acta Biomater 2012;8:677-685. Magalon J, Bausset O, Serratrice N, et al. Characterization and comparison of 5 platelet-rich plasma preparations in a single-donor model. Arthroscopy 2014;30:629-638. Krüger JP, Endres M, Neumann K, Haupl T, Erggelet C, Kaps C. Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid. J of Orthop Res 2010;28:819-827. Pereira H, Frias AM, Oliveira JM, Espregueira-Mendes J, Reis RL. Tissue engineering and regenerative medicine strategies in meniscus lesions. Arthroscopy 2011;27: 1706-1719. Gonzales VK, de Mulder EL, de Boer T, et al. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures. Tissue Eng Part C Methods 2013;19:892-899. Krüger JP, Hondke S, Endres M, Pruss A, Siclari A, Kaps C. Human platelet-rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells. J Orthop Res 2012;30:845-852. Giusti I, D’Ascenzo S, Manco A, et al. Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro. Biomed Res Int 2014;2014:630870. Riera KM, Rothfusz NE, Wilusz RE, Weinberg JB, Guilak F, McNulty AL. Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: Influences on meniscal cell proliferation and migration. Arthritis Res Ther 2011;13: R187. Kalwitz G, Andreas K, Endres M, et al. Chemokine profile of human serum from whole blood: Migratory effects of CXCL-10 and CXCL-11 on human mesenchymal stem cells. Connect Tissue Res 2010;51:113-122. Esparza R, Gortazar AR, Forriol F. Cell study of the three areas of the meniscus: Effect of growth factors in an experimental model in sheep. J Orthop Res 2012;10: 1647-1651. Zellner J, Hierl K, Mueller M, et al. Stem cell-based tissueengineering for treatment of meniscal tears in the avascular zone. J Biomed Mater Res B Appl Biomater 2013;101: 1133-1142.
Purpose: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. Methods: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. Results: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P ¼ .0001, 5% PRP: P ¼ .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P ¼ .0001) and 5% PRP (P ¼ .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P ¼ .0002, ACP: P ¼ .0147), cartilage oligomeric matrix protein (HS: P ¼ .0002, ACP: P ¼ .0005), and biglycan (HS: P ¼ .0002, ACP: P ¼ .0003), whereas PRP showed no inducing effect. Conclusions: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. Clinical Relevance: Our findings may suggest that HS might be a beneficial augment for meniscus repair.
M
enisci play a key role in the knee joint in terms of force transmission, shock absorption, provision of joint stability, and lubrication. Disruption and loss of the fibrous meniscus tissue increases the contact From the TransTissue Technologies GmbH, Department of Research & Development (U.F., J-P.K., G.H., M.E., C.K.), Berlin, Germany; Clinic for Traumatic Surgery and Orthopedics, Martin-Luther-Hospital (S.M., W.P.), Berlin, Germany; DeSimone Laboratory, Department of Cell Biology, University of Virginia (G.H.), Charlottesville, Virginia, U.S.A.; and Tissue Engineering Laboratory, Department of Rheumatology and Immunology, Charité - University Hospital Berlin (M.E.), Berlin, Germany. The authors report the following potential conflict of interest or source of funding: U.F., J-P.K., and M.E. receive support from TransTissue Technologies GmbH. W.P. receives support from Clinical Excellence Circle, Otto Bock Health Care, Karl Storz Endoscopy, and AAP Implants. C.K. receives support from TransTissue Technologies GmbH and BioTissue AG. Received January 30, 2015; accepted November 17, 2015. Address correspondence to Undine Freymann, Ph.D., TransTissue Technologies GmbH, Charitéplatz 1, 10117 Berlin, Germany. E-mail: undine. [email protected] Ó 2016 by the Arthroscopy Association of North America 0749-8063/1591/$36.00 http://dx.doi.org/10.1016/j.arthro.2015.11.033
pressure on the articular cartilage and can cause longterm articular cartilage injuries followed by dysfunction and secondary osteoarthritis.1 Annually, more than 1.5 million meniscus procedures are performed across the United States and Europe that most often include partial meniscectomy for irreparable meniscus lesions.2 In recent years, there has been a growing interest in using regenerative medicine approaches to repair damaged meniscus tissue.3 Thereby, optimal healing may rely on (1) a mechanical scaffold for cell adherence and for supporting new tissue formation, (2) the appropriate cell type to initiate tissue healing and new matrix synthesis, and (3) signals to stimulate cell differentiation and expression of matrix genes.4,5 Bloodderived products are considered as an attractive autologous source of such stimulatory signals. Consequently, blood-derived products such as human serum (HS)6 and different types of platelet concentrates7,8 have been used in vitro and in vivo to support mesenchymal tissue regeneration.
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The migration and differentiation of cells may be mediated by cytokines and growth factors. Recent in vitro studies showed a recruiting and differentiating effect of HS on human meniscus cells and meniscus-like matrix formation in high-density cultures as well as in 3-dimensional polyglycolic acid/hyaluronan scaffolds.9,10 Platelet-rich plasma (PRP) preparations are defined as suspensions of platelets in plasma, characterized by a platelet concentration that is higher than that of the original blood.11 In brief, platelets, once activated, release growth factors from their alphagranules that play an important role in the healing process and in the formation of different tissue types.7 Thus, they are also an attractive tool for the treatment of meniscal lesions. Different types of platelet concentrates can be prepared either by centrifugation methods or by commercially available separation kits (e.g., Autologous Conditioned Plasma [ACP]), which are designed for direct intraoperative use. ACP is prepared by a separation kit and described as a low-concentrate leukocyte-poor platelet preparation in contrast to those prepared by standard centrifugation methods that are characterized by a high amount of platelets and a low leukocyte content (called PRP in this study).11 Aiming at a cell-free scaffold-based approach for clinical meniscus repair, we follow the route that such a repair attempt may be improved by cell recruitment followed by cell growth and meniscal extracellular matrix (ECM) formation. Therefore, the purpose of this study was to evaluate the effect of 10% HS, 5% PRP, and 5% ACP on migration, proliferation, and ECM synthesis of human meniscus cells. We hypothesized that, because of the different growth factor content of HS, PRP, and ACP, these 3 blood-derived products might show different stimulatory potentials for cell migration, proliferation, and ECM formation of human meniscus cells.
Methods Preparation of Human Meniscus Cells, Human Serum, and Platelet Concentrates Patients (male or female; >18 years; n ¼ 5; Department of Orthopedics, Martin-Luther Hospital Berlin, Germany) scheduled for total knee arthroplasty (TKA) or postmortem donors (male or female; >18 years; n ¼ 5; Department of Pathology, Charité - University Hospital Berlin, Germany) were included. From each patient/donor, 1 lateral meniscus with macroscopic signs of degeneration such as tissue defibration and/or fatty degeneration was used. Menisci from postmortem donors were resected within 24 hours after death. Mensici were transferred immediately to the laboratory in Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) containing 100 U/ml penicillin and 100 mg/ml streptomycin (1% pen/strep;
Biochrom) to perform cell isolation. Human meniscus cells were isolated by enzymatic digestion in DMEM containing 1% pen/strep, 1% gentamycin, 0.04% amphotericin B (all Biochrom), 10% HS (DRK Blutspendedienst, Berlin, Germany), 10,000 U collagenase II (Biochrom), and 45 U collagenase P (Roche, Basel, Switzerland) for 18 to 20 hours in an incubator (37 C, 95% humidity, and 5% CO2). Cells were cryopreserved in passage 0. To obtain the required cell number (46 106 cells for pellet cultures, 12 106 cells for proliferation assay) and to ensure comparable experimental conditions (same cell pool), cells had to be expanded. For preparation of the cell pools, cells were thawed, seeded at a cell density of 10,000 cells/cm2, and cultured up to passage 3 in DMEM containing 1% pen/ strep and 10% HS. The study was approved by the ethical review board of the Charité - University Hospital Berlin. HS was prepared from 450 ml whole blood samples without anticoagulant of 10 healthy, anonymous blood donors aged between 18 and 65 years (DRK Blutspendedienst). After coagulation, the serum fraction (approximately 200 ml) was separated from the blood clot and residual blood cells and fibrinogen were removed by additional centrifugation (4,400 rpm for 10 minutes). Then, HS was heat-inactivated for 30 minutes at 56 C in a water bath. Ten individual HS preparations were pooled for further experiments. PRP (high-concentrate leukocyte-poor platelet preparation) is a licensed drug (Thrombocyte Concentrate N-W, DRK Blutspendedienst), which was prepared by centrifugation methods. First initial centrifugation of whole blood from healthy, anonymous blood donors aged between 18 and 65 years was performed to remove cellular elements from the plasma. Thereby, 90% to 95% of platelets were concentrated along with the leukocytes in the buffy coat layer above the tightly packed erythrocyte infranatant. During further processing, the platelet-enriched buffy coat was separated and resuspended in a platelet additive solution. Four buffy coats were pooled on the day after collection and centrifuged to remove contaminating leukocytes and erythrocytes. Pooled buffy coat-derived platelet concentrates were passed through a leukoreduction filter to reduce the residual leukocytes to less than 1 106 cells. The final platelet preparation (PRP) contained functional intact and high-concentrated platelets (3.2 1011 platelets) as an active agent as specified by the manufacturer. For our studies, 3 of these PRP preparations were pooled in equal amounts before use. ACP (low-concentrate leukocyte-poor platelet preparation) was prepared by a commercially available separation kit (ACP Double Syringe System, Arthrex, Munich, Germany) from 3 healthy blood donors (age 27 to 35 years, 2 males, 1 female). Per donor, a 15 ml venous blood sample was collected slowly in a special
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
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double syringe provided by the manufacturer. Blood was mixed in the syringe with 1 ml of anticoagulant citrate dextrose-A (Sigma-Aldrich, Darmstadt, Germany) to prevent anticoagulation. The double syringe was placed in 1 bucket of a customized centrifuge and a single centrifugation was performed (1,500 rpm for 5 minutes). At the end, the supernatant ACP (3 to 4 ml) was transferred from the larger outer syringe into the small inner syringe, carefully avoiding mixing. The preparation procedure was performed 3 times per donor to increase the final ACP volume per donor. For further experiments, ACP preparations from 3 individual blood donors were pooled in equal amounts before use. All pools of blood-derived products (HS, PRP, ACP) were stored at 20 C and thawed before use. PRP and ACP pools were thawed slowly at 4 C, followed by centrifugation at 4 C at 1,600 g for 10 minutes to remove residual fibrinogen. Supernatants were used immediately for further experiments.
as control media. After 24 hours, the medium was replaced by DMEM containing ITSþ1 supplemented with either 10% HS, 5% PRP, or 5% ACP (n ¼ 3 per time point and experimental group) and cells were maintained for up to 9 days. Concentrations of tested blood-derived products were chosen on the basis of the results from chemotaxis assays and previous studies.9 For DNA analysis, harvested cells were incubated with 1 ml papain-cystein hydrochloride (0.125 mg/ml in distilled water) for 16 hours at 60 C. The supernatant was stored at 20 C. Samples were diluted (1:2 to 1:8, v/v) in 2 M sodium chloride and 0.05 mM sodium hydrogen phosphate (all Sigma-Aldrich). Serial dilutions of calf thymus DNA (Life Technologies, Darmstadt, Germany) were used to prepare a standard curve and to calculate the DNA content of samples. One hundred microliters of standard or sample was mixed with 100 ml of 0.67 mg/ml bisbenzimide (Life Technologies) and measured at 360 nm with light emission at 460 nm.
Cell Migration Assays The chemotactic effect of HS, PRP, and ACP on human meniscus cells (cell pool of 5 postmortem donors, 3 104 cells/well) was analyzed using an 8-mm-pore polycarbonate membrane assay (96-well ChemoTx, Neuro Probe, Gaithersburg, MD) according to the manufacturer’s recommendations. Concentrations between 1% and 10% of PRP and ACP were analyzed each in biological triplicates for 3 individual blood donors/preparations. Migratory activity of cells toward HS was tested only for 10% HS. The migratory effect of rising concentrations of HS (0.1% to 100%) has been reported previously and showed that 10% HS recruited the most human meniscus cells.9 Different concentrations of blood-derived products were prepared by dilution in DMEM (low glucose) containing 0.1% HS. This medium also served as control. After 20 hours at 37 C, cells that attached underneath the polycarbonate membrane were fixed with acetone/methanol 1:1 (v:v) for 3 minutes and counterstained with Hemacolor Rapid Stain (Merck, Darmstadt, Germany). Cells that migrated through the filter pores were analyzed microscopically. Pictures were taken from 3 representative visual fields of each well, stained cells were counted using ImageJ (National Institute of Health, Bethesda, MD), and the cell number was extrapolated to the size of the well.
Studies on Meniscal Extracellular Matrix Formation The influence of HS, PRP, and ACP on ECM formation was evaluated in a standard high-density pellet assay (cell pool of 5 TKA patients, 250,000 cells/pellet), as described previously.9 ECM formation was induced by adding either 10% HS, 5% PRP, or 5% ACP to DMEM (high glucose) containing 1% ITSþ1, 1 mM sodium pyruvate, 0.35 mM L-proline, 0.17 mM Lascorbic acid-2-phosphate, and 0.1 mM dexamethasone (all Sigma-Aldrich), 1% pen/strep and 2% 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (Biochrom). Pellets cultured in complete DMEM without PRP, ACP, or HS served as controls. Fifty-two pellets were prepared for each group (PRP, ACP, HS, control). The medium was exchanged every 2 to 3 days and cells were maintained for up to 28 days. For histological evaluation of ECM deposition, pellets were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA), and frozen and cryo-slides (6 mm) were prepared. Avascular tissue samples of human meniscus, which have been prepared from the original degenerative donor tissue before cell isolation, served as positive control. H&E staining was performed to assess pellet and cell morphology. Proteoglycans were stained with Alcian Blue 8GX (Carl Roth, Karlsruhe, Germany), followed by counterstaining with nuclear fast red (SigmaAldrich). For collagen type I and type II stainings, cryoslides were incubated for 40 minutes with mouse antihuman type I or rabbit antihuman type II collagen antibodies (Acris, Herford, Germany). Mouse and rabbit immunoglobulin G served as control (DAKO, Hamburg, Germany). Detection was performed using the EnVisionþþ System HRP Kit (DAKO), followed by counterstaining with hematoxylin. For each staining,
Cell Proliferation Studies The effect of HS, PRP, and ACP on cell proliferation was evaluated using a pool of meniscal cells (cell pool of 5 TKA patients). Cells were seeded in T25 cell culture flasks (10,000 cells/cm2) in DMEM (low glucose) containing 1% insulin-transferrin-sodium selenite with linoleic acid (ITSþ1; Sigma Aldrich), which also served
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n ¼ 3 pellets per group and n ¼ 3 slides per pellet were analyzed. Histomorphometric analysis was performed by 1 observer using the Adobe Photoshop Software as described.12 In brief, a standard color was defined that represents the particular color of the specific staining. The amount of stained pixels in relation to the total amount of pixels of the section represents the percentage of the positively stained area. Gene Expression Analysis Expression levels of collagen type I and II, aggrecan, biglycan, cartilage oligomeric protein (COMP), and biglycan, which have been shown to be expressed in native meniscus tissue,10 were examined in pellets at early day 14 (pool of n ¼ 20 pellets per experimental group). Total RNA was isolated and 590 ng was transcribed into single strand cDNA using the iScript Kit (BIO-RAD Laboratories, Munich, Germany) according to the manufacturer’s recommendations. Real-time reverse transcriptase polymerase chain reaction was performed using the “SYBR Green PCR Core Kit” (Life Technologies) and a PCR cycler (iCycler, BIO-RAD). The relative expression of glyceraldehyde-3-phosphate dehydrogenase was used to normalize the cDNA samples. The fold change (FC) of expression values for a given marker gene per group was calculated in relation to controls.
Table 1. Patient and Donor Characteristics Cell Pool (n ¼ 5) 1
2
66 68 77 78 79 48 85 56 60 69
Age, yr mean 73.6
mean 63.6
Sex (Male/ Female) f f f m m f f m m m
f, female; m, male.
Statistical Analysis Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH, Erkrath, Germany). The Kolmogorov-Smirnov method was used to determine normal distribution and equal variance of data. Subsequently, 1-way analysis of variance and multiple comparison procedure according to the StudentNewman-Keuls method were used for statistical analysis of data within 1 group as well as for intergroup analysis. Significant differences were considered at P < .05. For gene expression analysis, differential expression was considered at an FC of less than 2 or greater than 2 and P < .05. Bars present the mean value of data with standard deviation.
Results Patient and Donor Characteristics Patient and donor characteristics are given in Table 1. Chemotactic Effect of HS, PRP, and ACP on Human Meniscus Cells All blood-derived preparations showed a migratory effect on human meniscus cells (Fig 1A). Stimulation with 10% HS significantly recruited meniscus cells, 13,308 25 cells (mean standard deviation) compared with nonstimulated controls (5,388 1,254 spontaneously migrating cells). Stimulation with 1% PRP (10,142 2,755 cells) to 10% PRP (17,861
Fig 1. Migratory effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on human meniscus cells. (A) Migratory effects have been quantified by enumerating the number of migrating cells on stimulation with HS, ACP, and PRP. All 3 blood-derived products increase, to different extents, the number of migrating human meniscus cells. (B) Intergroup comparison between 10% HS, 5% PRP, 5% ACP, and nonstimulated controls. Asterisks (*) indicate significant differences (P > .05). The bars present the mean value of data with standard deviation (pool of n ¼ 5 donors) of each group.
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2,312 cells) showed significantly increased cell numbers when compared with controls and significant differences in migrating cells between PRP concentrations of 1% to 10%. Migratory activity significantly increased on stimulation with 1% ACP (11,821 1,305 cells) to 10% ACP (11,417 2,963 cells) when compared with controls, but did not show a dose-dependent increase in the number of migrating cells. Further intergroup comparison (Fig 1B) showed significantly increased cell numbers after stimulation with 5% PRP, 5% ACP, and 10% HS in comparison with nonstimulated controls. Therein, stimulation with 5% PRP led to the highest migratory activity of cells and showed significantly increased cell numbers in comparison with the 10% HS and 5% ACP group as well. Proliferative Effect of HS, PRP, and ACP on Human Meniscus Cells Cell proliferation was evident after stimulation of human meniscus cells with 10% HS, 5% PRP, and 5% ACP as well as in nonstimulated controls as shown by the increase of DNA content (Fig 2A). The DNA content significantly (P < .05) increased over cultivation time (day 0 to 9) within the HS group (1.60 0.06 to 6.08 0.12 mg/ml) and PRP group (1.56 0.23 to 5.91 0.12 mg/ml). Stimulation with 5% ACP significantly increased DNA values at day 6 (2.71 0.35 mg/ml) and day 9 (3.04 0.12 mg/ml) when compared with day 0. In nonstimulated controls, the DNA content significantly increased at day 6 (2.42 0.99 mg/ml) and at day 9 (3.04 0.35 mg/ml) when compared with the values at day 0. At day 9 (Fig 2B), the DNA content significantly increased in cultures stimulated with HS (6.08 0.12 mg/ml) and PRP (5.91 0.12 mg/ml) when compared with the ACP group (3.04 0.12 mg/ml) or with the nonstimulated control group (3.04 0.35 mg/ ml). In the ACP group, no proliferative stimulus could be detected after 9 days. Effects of HS, PRP, and ACP on Extracellular Matrix Formation All 4 groups of human meniscus pellet cultures (10% HS, 5% ACP, 5% PRP, control) showed ECM formation at day 28 (Fig 3). The morphology and dimensions of the pellets are presented in Figure 3A. Therein, nonstimulated controls showed a whiter and more oval appearance of the pellets compared with the other groups. Stimulation with PRP or ACP resulted in an enlargement of pellets compared with the round compact form of HS-stimulated pellets. Largest, but also most inhomogeneous, pellets were found on stimulation of cell pellets with ACP, followed by the PRP group. Evaluation of ECM formation by H&E staining (Fig 3B) showed an ECM rich in cells in nonstimulated controls. Pellets stimulated with HS developed a well-structured
5
ECM, rich in homogeneously distributed cells. PRPstimulated pellets formed an ECM with a cell-rich and compact part (Fig 3B, double black arrowhead) but also unstructured parts, poor in cells (Fig 3B, asterisk). ACPstimulated pellet cultures formed an ECM with a small and compact core rich in cells (Fig 3B, black arrowhead), surrounded by high amounts of unstructured ECM poor in cells (Fig 3B, asterisk). Histological staining of ECM components at day 28 (Fig 4) revealed that high-density pellets stimulated with HS developed a meniscus-like ECM with deposition of collagen type I, proteoglycans, and collagen type II (Fig 4 D-F) similar to findings in native meniscus tissue sections of the same donors (Fig 4 M-O). Pellets treated with PRP (Fig 4 G-I) or ACP (Fig 4 J-L) showed the formation of a fibrous and inhomogeneous ECM with weak deposition of collagen type I, proteoglycans, and collagen type II, located especially in the cell-rich ECM area and not in the surrounding fibrous parts. Nonstimulated controls (Fig 4 A-C) also showed the deposition of collagen type I and faint stainings of proteoglycans and collagen type II. Histomorphometric quantification at day 28 (Table 2) showed significant differences in collagen type I stained areas between nonstimulated controls and all other 3 stimulated groups as well as a significant decreased deposition of collagen type I in ACP-stimulated pellets in comparison with the HS and PRP groups. Histomorphometric quantification of proteoglycans (alcian blue staining) showed a significant increase in HSstimulated pellets when compared with controls, with the ACP group, and with the PRP group. ACP- and PRPstimulated pellets showed significant less proteoglycans than nonstimulated controls. The HS group showed the highest and the ACP group the weakest deposition of proteoglycans among all groups. Quantification of collagen type II stained areas showed a significant increase in HS-stimulated pellets when compared with the ACP and PRP groups. ACP- and PRP-stimulated pellets showed significant less collagen type II deposition than nonstimulated controls. Among all groups, HS-stimulated pellets showed the highest and ACPstimulated pellets the weakest deposition of collagen type II. Gene Expression Analysis of Human Meniscus Pellet Cultures To assess the effects of HS, PRP, and ACP on gene expression level, analysis of meniscus-related genes was performed at early day 14 (Fig 5). Stimulation of pellets with HS significantly induced the expression of aggrecan (FC ¼ 8.9 1.13), COMP (FC ¼ 6.07 0.73), and biglycan (FC ¼ 2.71 0.26) at day 14 compared with controls and also led to significantly elevated aggrecan and COMP expression levels in comparison with ACPand PRP-stimulated pellets. Stimulation of pellets with
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Fig 2. Proliferative effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on human meniscus cells. (A) Proliferative effects have been evaluated by measuring the content of genomic DNA of cells on stimulation with HS, PRP, and ACP for up to 9 days. DNA content and thus the number of progenitor cells increased after stimulation with HS and PRP. Nonstimulated controls and cells stimulated with ACP showed a comparable DNA content. (B) Intergroup comparison between all groups at day 9. Asterisks (*) indicate significant differences (P > .05). The bars present the mean value of data with standard deviation (pool of n ¼ 5 donors) of each group.
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Fig 3. Effect of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on meniscal extracellular matrix (ECM) formation at day 28. (A) Morphology and dimension of human meniscus pellet cultures stimulated with HS, PRP, or ACP, and nonstimulated controls. (B) H&E staining. Stimulation with HS showed formation of compact pellets with homogeneous and cell-rich ECM, whereas stimulation with ACP resulted in a large inhomogeneous pellet with a small area of cell-rich ECM (black arrowhead) surrounded by an unstructured, fibrous, and cell-poor ECM in the outer zone. PRP-stimulated pellet cultures showed a compact ECM area rich in cells (double black arrowhead), surrounded by unstructured fibrous ECM parts (asterisks).
ACP significantly induced the expression of aggrecan (FC ¼ 2.87 0.39), COMP (FC ¼ 3.49 0.35), and biglycan (FC ¼ 2.12 0.15) at day 14 compared with controls, but to a lesser extent than HS. PRP showed no effect on gene expression levels of aggrecan, COMP, or biglycan and led to a significantly decreased expression of collagen type I (FC ¼ 0.37 0.05) at day 14 compared with controls. Collagen type II (data not shown) and collagen type I expression levels were not induced by stimulation of pellets with HS, PRP, or ACP at early day 14 compared with nonstimulated controls.
Discussion In the current study, blood-derived products such as HS, PRP, and ACP enhanced cell migration, whereas cell proliferation was significantly increased only by HS and PRP. Gene expression analyses of pellet cultures at day 14 revealed an induction of genes coding for large and small meniscal proteoglycans like aggrecan and biglycan and other ECM proteins like COMP after stimulation with HS and ACP, although to a lesser extent. PRP did not show an inducing effect on fibrochondrogenic marker gene expression. An induction of collagen type I and type II expression was not detectable at early day 14 in any stimulated group. Despite that, there is evidence that gene transcription and protein translation occurred to a later time point, because we histologically evaluated the deposition of collagen type I and type II at
day 28. Formation of a well-structured meniscus-like ECM was stimulated only by HS, whereas PRP and ACP led to the formation of an unstructured and more fibrous ECM. The proliferative effect of PRP and HS on human meniscus cells is in line with other in vitro studies, showing a positive mitogenic effect on monolayer rabbit meniscal cells,13 sheep meniscal cell cultures,2 or human meniscus cells.10,14 Besides the impact on cell proliferation, all 3 bloodderived products have been shown to significantly stimulate the migration of human meniscus cells. The chemotactic effect of platelet concentrates is comparable with its effect on human subchondral mesenchymal progenitor cells15 or human tenocytes.16 The chemotactic effect of HS on human meniscus cells has been reported previously, where doses between 1% and 20% HS effectively attracted human meniscus cells,9 and is confirmed again by this study for 10% HS. Our results are comparable with other studies, which showed the effects of fetal bovine serum on cell migration and proliferation of porcine meniscus cells in micro-wound and repair models.17 The effect of HS may be due to its protein composition including cytokines, growth factors, and/or diverse chemokines, which have been shown to stimulate chemotaxis of cells.18 Also various growth factors have been proven to be effective in meniscus in vitro studies (Table 3).
8
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Fig 4. Histological and immunohistochemical evaluation of meniscal extracellular matrix (ECM) formation in human meniscus pellet cultures at day 28. Effects of human serum (HS), platelet-rich plasma (PRP), and autologous conditioned plasma (ACP) on ECM formation have been evaluated by histological and immunohistochemical analyses. Alcian blue and collagen type I and type II staining showed that nonstimulated controls showed high deposition of (A) collagen type I and faint stainings for (B) proteoglycans and (C) collagen type II. Cells stimulated with HS formed a meniscus-like ECM with high deposition of (D) collagen type I, formation of (E) proteoglycans, and high deposition of (F) collagen type II. (G-L) Cells stimulated with PRP or ACP showed inhomogeneous ECM formation. Therein, deposition of (H, K) proteoglycans and collagen (G, J) type I and (I, L) type II was found only in nonfibrous ECM areas. (M-O) Stained sections of the inner part of the original degenerative meniscus tissue samples served as positive control. Tissue samples showed homogenously distributed cells and the presence of (N) proteoglycans and collagen (M) type I and (O) type II.
9
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
Table 2. Analysis of Histomorphometric Data (Day 28) Using 1-Way Analysis of Variance and Multiple Comparison Procedure According to the Student-Newman-Keuls Method Comparison Group 1/Group 2 Collagen type I stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control Alcian blue stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control Collagen type II stained area HS/Control HS/PRP HS/ACP PRP/Control PRP/ACP ACP/Control
Mean SD (Group 1)/Mean SD (Group 2)
P Value
Significance (P < .05)
57.0 57.0 57.0 42.7 42.7 19.6
21.6/76.9 21.6/42.7 21.6/19.6 19.2/76.9 19.2/19.6 11.9/76.9
4.1 19.2 11.9 4.1 11.9 4.1
.0170 .0721 .0002 .0004 .0043 .0002
Yes No Yes Yes Yes Yes
57.4 57.4 57.4 26.1 26.1 6.1
10.4/43.3 11.0 10.4/26.1 9.9 10.4/6.1 5.9 9.9/43.3 11.0 9.9/6.1 5.9 5.9/43.3 11.0
.0049 .0001 .0002 .0018 .0005 .0001
Yes Yes Yes Yes Yes Yes
79.7 79.7 79.7 30.2 30.2 16.8
18.7/67.5 31.2 18.7/30.2 13.5 18.7/16.8 6.4 13.5/67.5 31.2 13.5/16.8 6.4 6.4/67.5 31.2
.2419 .0002 .0002 .0001 .1737 .0002
No Yes Yes Yes No Yes
ACP, autologous conditioned plasma; HS, human serum; PRP, platelet-rich plasma; SD, standard deviation.
The effects of HS, PRP, and ACP on gene expression and meniscus-like ECM formation are differing. Stimulation of meniscus cells with HS induced meniscus-related genes (aggrecan, biglycan, COMP) and led to the formation of a homogeneous meniscal ECM. Results are in line with previous studies, which showed a fibrocartilaginous gene expression profile and meniscus-like ECM formation after stimulation of human meniscus cells with HS in pellet and scaffold cultures.9,10 Small and large proteoglycans like biglycan and aggrecan have been stated to resist compressive and tensile forces in the adult human meniscus.10 Thus, the use of HS with its proven stimulatory potential to induce expression and deposition of meniscal ECM in vitro might also improve meniscus repair approaches in vivo. Platelet concentrates like PRP and ACP could not lead to comparable results on protein or gene expression level. Stimulation with ACP led to an early gene induction of aggrecan, biglycan, and COMP, but finally only resulted in the formation of an inhomogeneous and nonmeniscal ECM after 28 days. Stimulation with PRP led to the formation of a better structured, but still more fibrous ECM and showed no induction of fibrochondrogenic genes. Gene expression results for platelet preparations are comparable with those reported by others, where mRNA expressions have been measured after monolayer cultivation of rabbit meniscus cells with 10% PRP.13 However, in the same report, a gelatin gel immersed with PRP effectively repaired full-thickness meniscal defects in the rabbit model.13 In contrast to that, no significant
improvement was found when platelet preparations were used with a hyaluronan-collagen composite matrix for the treatment of rabbit meniscal defects.20 Obviously, further studies are needed to elucidate whether platelet preparations like PRP or ACP are beneficial for meniscus regeneration. In summary, HS and platelet concentrates like PRP and ACP as a source of growth factors showed different potentials to stimulate migration, proliferation as well as meniscus-like ECM formation of human meniscus cell in vitro. Limitations The current study has some limitations. This in vitro study does not reflect the in vivo situation. Therefore, our in vitro study cannot predict the effects or activities of blood-derived products like HS, PRP, or ACP in vivo. All studies were performed using a pool of 5 individually cell preparations and with pools of blood-derived products. Because of limited donor tissue availability, we used degenerative whole meniscus tissue derived from old donors with age-related degenerative changes, which do not represent the typical younger patient cohort for meniscus repair or replacement. We also did not discriminate between degenerative status and distinct meniscus regions. On the basis of the results of our migration experiments, we tested only a singular concentration for studies on proliferation and ECM formation and did not evaluate differential effects of different doses. Our study is also limited by only 1 reviewer for evaluation of histological findings and by a low sample size in some groups and varying blood
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Fig 5. Real-time gene expression analysis of meniscus marker genes at day 14. Fold change analysis of meniscal marker genes like collagen type I, aggrecan, cartilage oligomeric protein (COMP), biglycan, and collagen type II expressed in human meniscus pellet cultures compared with nonstimulated controls. Asterisks represent significant (P < .05) differences in gene expression between all 4 groups (human serum [HS], platelet-rich plasma [PRP] or autologous conditioned plasma [ACP], and nonstimulated controls). The bars show the mean fold change (n ¼ 3; pool of n ¼ 20 pellets per experimental group) of each group and standard deviation is plotted.
donors among the groups. The study would have been strengthened by using preparations derived from blood drawn of the same donors to probably elicit different biological activities of the different blood-derived products.
Conclusions Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of
Table 3. Growth Factor Application in Meniscus In Vitro Studies Growth Factor
Abbreviation
Study Outcome: In Vitro Effect of Growth Factor
Reference
Platelet-derived growth factor Transforming growth factor-beta 1 Fibroblast growth factor
PDGF TGF-ß1 FGF
Moran et al.2 (review)
Insulin-like growth factor
IGF
Vascular endothelial growth factor
VEGF
Ovine meniscus 3D scaffold cultures: stimulation of cell proliferation and migration, increase in collagen type I expression, decrease in collagen type II production Ovine meniscus 3D scaffold cultures: stimulation of cell proliferation, increase in collagen type I expression Ovine/bovine meniscal explant model: increase in DNA and extracellular matrix synthesis and induction of cell migration Ovine meniscus cells from the avascular zone: stimulation of collagen type I and type II synthesis
3D, 3-dimensional.
Moran et al.2 (review)
Esparza et al.19 2012
MENISCUS REPAIR AND BLOOD-DERIVED PRODUCTS
nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches.
10.
Acknowledgment The authors thank Samuel Vetterlein and AnneCathrin Behr for excellent technical and laboratory assistance. This study was supported by the Bundesministerium für Wirtschaft und Technologie, project “Zentrales Innovationsprogramm Mittelstand,” grant ID: KF2699502FR2.
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