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Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog George A. Ballard a,1, Jennifer J. Warnock a,*, Gerd Bobe b, Katja F. Duesterdieck-Zellmer a, Lindsay Baker a,2, Wendy I. Baltzer a, Jesse Ott a a b

College of Veterinary Medicine, Oregon State University, 105 Magruder Hall, 700 SW 30th St., Corvallis, OR 97331, USA Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science Center, Corvallis, OR 97331, USA

A R T I C L E

I N F O

Article history: Received 20 October 2013 Accepted 4 May 2014 Keywords: Tissue engineering Meniscus Cell culture Synovium Osteoarthritis

A B S T R A C T

Tissue engineering is a promising field of study toward curing the meniscal deficient stifle; however the ideal cell type for this task is not known. We describe here the extraction of synoviocytes and meniscal fibrochondrocytes from arthroscopic debris from six dogs, which were cultured as tensioned bioscaffolds to synthesize meniscal-like fibrocartilage sheets. Despite the diseased status of the original tissues, synoviocytes and meniscal fibrochondrocytes had high viability at the time of removal from the joint. Glycosaminoglycan and collagen content of bioscaffolds did not differ. Meniscal fibrochondrocyte bioscaffolds contained more type II collagen, but collagen deposition was disorganized, with only 30–40% of cells viable. The collagen of synoviocyte bioscaffolds was organized into sheets and bands and 80–90% of cells were viable. Autologous, diseased meniscal fibrochondrocytes and synoviocytes are plausible cell sources for future meniscal tissue engineering research, however cell viability of meniscal fibrochondrocytes in the tensioned bioscaffolds was low. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The canine stifle menisci are c-shaped fibrocartilages which provide major weight-bearing functions, including proprioception (Zimny, 1988; Zimny et al., 1988) joint lubrication, (Clark et al., 1999) shock absorption, (Voloshin and Wosk, 1983) relief of femoral– tibial incongruity (Mow, 1992) load transmission, (Ahmed, 1983) and joint stability (Levy et al., 1982; Voloshin and Wosk, 1983). Meniscal extracellular matrix is composed primarily of type I collagen organized into circumferential bands bound by radial tie fibers (Fithian et al., 1990; Kambic and McDevitt, 2005). The menisci also contain type II collagen, located primarily in the axial region and around radial tie fibers (Eyre and Wu, 1983; Kambic and McDevitt, 2005) and glycosaminoglycans (GAG) (Adams and Ho, 1987; Nakano et al., 1997; Stephan et al., 1998), including aggrecan (Valiyaveettil et al., 2005). This functionally critical fibrocartilage has limited healing capabilities; in particular, the avascular, axial white–white zone does not heal spontaneously. (Arnoczky and Warren, 1983; Kobayashi et al., 2004). Therefore, avascular meniscal injuries are treated with

* Corresponding author. Tel.: +1 541 737 6859; fax: +1 541 737 4818. E-mail address: [email protected] (J.J. Warnock). 1 Current address: Peace River Veterinary Clinic, Punta Gorda, Florida, USA. 2 Current address: WestVet Specialty Center, Garden City, Idaho, USA.

partial meniscectomy, to provide short term relief from clinical signs such as painful joint locking and popping. Unfortunately, partial meniscectomy does not replace the critical weight bearing functions of the meniscus and hastens the development of secondary arthritis (Berjon et al., 1991; Connor et al., 2009; Cox et al., 1975) which is increased proportionally to the amount of meniscal tissue resected (Berjon et al., 1991). Tissue engineering through production of living replacement tissue may be one method for curing canine meniscal deficiency. Unfortunately, the ideal cell source and in vitro biomechanical and biological conditions for creating such a fibrocartilage implant have not been determined. The in vitro biomechanical environment may be particularly critical to the meniscal tissue engineering effort: application of biomechanical stimulation in vitro has a profound effect on extracellular matrix (ECM) formation due to the principles of mechanotransduction (Lavagnino and Arnoczky, 2005; Mauck et al., 2002). Type I collagen tends to form in tissues influenced by tensile forces, and GAG and type II collagen tend to form in tissues subjected to compressive forces (AufderHeide and Athanasiou, 2004; Benjamin and Ralphs, 1998; Kambic and McDevitt, 2005). To that end, the type I collagen of the abaxial 2/3 of the meniscus converts compressive weight bearing forces into tensile hoop strains, while the GAG and type II collagen of the axial meniscus sustain primarily compressive forces (Fithian et al., 1990; Mow et al., 1989). Thus successful tissue engineering of the meniscus will likely require a combination of tensile and compressive forces to induce functional ECM.

http://dx.doi.org/10.1016/j.rvsc.2014.05.002 0034-5288/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: George A. Ballard, et al., Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog, Research in Veterinary Science (2014), doi: 10.1016/j.rvsc.2014.05.002

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Synovium is a non-weight bearing tissue lining the joint capsule, which produces hyaluronic acid and lubricin (Lee et al., 2010). Synovium contains loosely organized types I, III, and VI collagen, glycosaminogycans, fibronectin, and vitronectin (Ando et al., 2007, 2008; Okada et al., 1990; Price et al., 1996). Despite the dramatic differences in matrix architecture and function, synovium has been extensively investigated for meniscal tissue engineering and has been concluded to hold tremendous promise for this purpose (Fox and Warnock, 2011). Autologous, osteoarthritic-joint origin synovium has been investigated as a cell source for fibrocartilage tissue engineering in dogs because of its ease of harvest, and in vitro (Warnock et al., 2011, 2012) and in vivo (Tienen et al., 2006) ability to synthesize fibrocartilage ECM. Additionally, canine synovium gathered during standard arthroscopic partial synovectomy can be used to synthesize collagenous tensioned bioscaffolds (Warnock et al., 2013). Use of normal synovium as a cell source would require violation of a healthy, unaffected joint, which is clinically undesirable. Healthy animal meniscal fibrochondrocytes have been used in meniscal tissue engineering research (Hoben et al., 2007; Tan et al., 2010); however, they also represent a poor cell source option for clinical application, requiring injury or sacrifice of a patient’s unaffected meniscus. Axial meniscal tissue removed during partial meniscectomy is a logical source of autologous cells for reforming axial meniscal fibrocartilage in vitro. However, the synthetic capability and cell viability of meniscal fibrochondrocytes from diseased, excised meniscal tissue are unknown. The long term goal of this research is to identify a viable and synthetically active cell source available for autologous meniscal tissue engineering purposes. Thus, the first objective of this study was to determine the viability and cell yield of autologous meniscal fibrochondrocytes obtained from arthroscopic meniscectomy debris in dogs, with the hypothesis that arthroscopic meniscectomy debris would yield fewer cells, with lower viability than arthroscopic synovectomy debris. Surgically implanted collagenous sheets help regenerate the meniscus if the lesion extends to the vascular red zone (Cook et al., 1999, 2006a, 2006b) but not in white zone avascular lesions (Welch et al., 2002). Conceivably, tissue engineering a surgical implant consisting of a collagenous sheet of living tissue, which contains the ECM components of the axial meniscus, and can remodel and adapt to the intra-articular environment, could be helpful in guiding regeneration of lost axial meniscal tissue. Thus, toward engineering such an implant, the second study objective was to synthesize living, collagenous sheets. The collagen and glycosaminoglycan (GAG) content of meniscal fibrochondrocyte hyperconfluent monolayer cell sheets (‘CS’) was compared to tensioned meniscal fibrochondrocyte bioscaffold sheets, to determine the effect of long term culture with tension. The hypothesis was that with culture under tension as tensioned meniscal fibrochondrocyte bioscaffolds (‘TMB’), meniscal fibrochdondrocytes would have higher GAG and collagen content relative to the meniscal CS, as has been determined in synoviocytes (Warnock et al., 2013). To compare the effect of cell type on collagenous sheet synthesis, cell viability and ECM formation were compared between meniscal fibrochondrocytes versus synoviocytes cultured as tensioned bioscaffolds (tensioned synoviocyte bioscaffold, or ‘TSB’). We hypothesized that meniscal-like ECM content and cell viability of TMB would be greater versus TSB.

for naturally occurring, chronic, non-contact cruciate ligament tears and medial meniscal bucket handle tears. Synovial villi which blocked the view of the cruciate ligaments and medial and lateral femoral condyles were arthroscopically resected as clinically required using a tissue shaver (Stryker, San Jose, CA) with a 3.5 mm aggressive shaver blade run at 1800 rpm, and were retained for culture as previously described (Warnock et al., 2012). All dogs had non-displaced, avascular medial meniscal bucket handle tears of the caudal body and horn. Torn tissue was removed by displacing the ‘bucket handle’ of meniscal tissue cranially with a hooked-tip arthroscopic probe (Arthrex, Naples FL); then the axial portion of the ‘handle’ at the meniscal body was released with a disposable arthroscopic push knife (Smith & Nephew, Inc., Andover MA); then the portion at the caudal meniscotibial ligament was released using an arthroscopic punch (Arthrex, Naples FL). The freed tissue fragments were removed using arthroscopic tissue graspers (Arthrex, Naples FL). Harvested synovial villi and meniscectomized tissue fragments were immediately placed in 50 ml polypropylene tubes containing 40 ml of Dulbeccos’ Modified Eagle’s Media (DMEM) with 10% fetal bovine serum (FBS), warmed to 37 °C and transported to the laboratory. Tubes containing synovial villi were centrifuged at 313 g and then media was decanted. Meniscal tissue was additionally sterily minced into 2–3 mm × 2–3 mm pieces using #10 Bard-Parker blade. Tissue fragments were transferred by pipette and/or sterile forceps into a digestion solution as will be described later. 2.2. Cell culture All tissues were completely digested with sterile type 1A clostridial collagenase, 10 mg/ml, in RPMI 1640 solution (Invitrogen) at 37 °C. Prior to transfer to culture flasks, a 20 µl sample of tissue digest was analyzed with the trypan blue exclusion assay to determine harvest viability and cell counts. Cells were cultured in monolayer at 37.8 °C, 5% CO2, 95% humidity with daily media changes consisting of DMEM supplemented with 17.7% FBS and other additives (‘SDMEM’; see Appendix). At the fourth passage (Han et al., 2010), cells were allowed to become hyperconfluent cell sheets, defined as cells overlapping each other in greater than 100% confluency. Upon spontaneous contraction off the flask floors, hyperconfluent cell sheets were removed from the flask in preparation for bioscaffold synthesis. One meniscal fibrochondrocyte CS from three of the six dogs were harvested upon reaching hyperconfluence. One meniscal fibrochondrocyte CS was also retained from the fourth dog for realtime reverse transcriptase PCR. To determine the effect of long term culture with tension, the double stranded DNA (dsDNA), GAG, and collagen quantity of CS were compared to that of tensioned meniscal fibrochondrocyte bioscaffolds (TMB). To compare the fibrochondrogenic potential of meniscal fibrochondrocytes versus synoviocytes, tensioned bioscaffolds were made from each cell type (TSB and TMB), using a previously described technique (Warnock et al., 2013). Briefly, hyperconfluent cell sheets were wrapped over 2.0 cm diameter, 22 ga cerclage wire hoops in three layers, with approximately 0.5 N of tension to avoid tearing. The TSB were placed in six-well plates in 9.0 ml of the supplemented DMEM described earlier, with the free end of the cell sheet facing down to prevent loosening. All TSB and TMB were harvested for analysis after a total of 30 days in culture (Ando et al., 2008; Tan et al., 2010).

2. Materials and methods 2.3. Tissue analyses 2.1. Tissue harvest With informed owner consent and Institutional Animal Care and Use Committee permission, synovial villi and meniscetomized debris were obtained from six dogs. Dogs received arthroscopy and TPLO

Tissue analyses examined the presence of ECM that is responsible for meniscal form and function, including type I collagen (Kambic and McDevitt, 2005), type II collagen (Kambic and McDevitt, 2005), α-smooth muscle actin (ASM) (Kambic et al., 2000; Spector,

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2001), and glycosaminoglycans (GAG) (Adams and Ho, 1987; Stephan et al., 1998), including aggrecan (Valiyaveettil et al., 2005). Expressions of inflammatory mediators interleukins 1(Il-1) and 6 (Il-6), tumor necrosis factor-alpha (TNFα), were investigated as these factors may be associated with decreased in vitro ECM synthesis in osteoarthritic joint-origin synoviocytes (Fiorito et al., 2005; Pei et al., 2008). 2.3.1. Histologic analysis Samples from each group were fixed in 10% buffered formalin for 48 h. After fixation, bioscaffolds were removed from their hoops using a #15 Bard-Parker blade. Samples were paraffin embedded, sectioned at 4–5 µm, and stained with Hematoxylin and Eosin, Masson’s trichrome, and Toluidine Blue. Immunohistochemistry was performed as previously described (Warnock et al., 2012) for type I collagen (rabbit #AB749P; 1:100 dilution; Millipore), type II collagen (rabbit #AB746P; 1:100; Millipore), and alpha smooth muscle actin (mouse #M0851; 1:30; Dako). Extracellular and intracellular immunoreactivity intensity and prevalence were rated as previously described (Wakshlag et al., 2011); immunoreactivity was localized to intracellular or extracellular staining, and ECM immunoreactivity was described as mild, moderate, or strong staining. As determined by hand count, intracellular immunoreactivity was categorized as positive in 50% of cells per 10× field, choosing three peripheral sites and three central sites. 2.3.2. Real-time RT-PCR Samples from each group were snap frozen in liquid nitrogen and stored at −80 °C. Quantitative real-time RT-PCR was performed as previously described (Chomczynski and Sacchi, 1986; Schmittgen and Livak, 2008) for Sry-type homeobox protein-9 (SOX-9), an embryonic chondrogenic transcription factor, collagen type I α1, collagen type 2 α1, aggrecan, Il-1β, Il-6,TNFα, and GAPDH (Appendix) using proprietary, pre-designed primers and probes (Taq-Man® Primers and Probes, Applied Biosystems Inc., Foster City, CA). Fold changes in gene expression were calculated using the following formula: fold change = 2−ΔΔCT = [(CTgene of interest − C T housekeeping geneGAPDH) TSB − (C T gene of interest − CThousekeeping geneGAPDH)TMB] (Table 1). 2.3.3. Tissue weight Samples from each group were lyophilized, dry weight obtained, digested, and used for double-stranded DNA, GAG, and collagen analysis (Warnock et al., 2012). 2.3.4. DNA quantification The Quant-iT PicoGreenTM (Invitrogen) double stranded DNA quantification assay was performed per manufacturer’s instructions; standard and sample fluorescence was read at 485 nm exci-

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tation/528 nm emission by a fluorometer (Quibit Fluorometer, Invitrogen). 2.3.5. Spectrophotometric extracellular matrix analyses The concentration of GAG was determined by the di-methylmethylene blue sulfated GAG assay (Farndale et al., 1986) using a spectrophotometer (Synergy HT–KC4, BioTec). Collagen content was determined by Erlich’s hydroxyproline assay (Reddy and Enwemeka, 1996). The hydroxyproline content was converted to collagen content using the following equation: (μg hydroxyproline × dilution factor)/ 0.13 = μg collagen (Ignat’eva et al., 2007), because hydroxyproline consists of approximately 13% of the amino acids in human meniscal collagen (Fithian et al., 1990; Fithian et al., 1990). The concentrations of GAG and collagen were standardized to tissue dry weight and expressed as percentage dry weight to allow comparison of the experimental neotissues to normal meniscal ECM content (Eyre and Wu, 1983). The chondrogenic index was calculated using the following equation: μg GAG/ug dsDNA (Li and Pei, 2011). The collagen index was calculated using the following equation: μg collagen/ ug dsDNA, to determine cellular GAG and collagen production, respectively. Total GAG and collagen content were also reported in μg/neotissue to allow comparison of total synthetic activity over the course of 30 days of in vitro culture between TSB and TMB. 2.3.6. Cell viability Samples from treatment groups 2 and 3 were washed three times in sterile phosphate buffered saline and immersed in 4 μM ethidium homodimer and 6 μ M acetomethoxy–calcein solution (Invitrogen), for 20 min at 37 °C, 5% CO2, 95% humidity. Hand counts of viable and non-viable cells per 10× objective field were made in five regions of the neotissue, using a laser microscope (Eclipse Tiu, Nikon). Due to the complex three-dimensional nature of the constructs, these cell counts provided an estimate of regional cell viability. 2.4. Statistical methods A D’Agostino & Pearson omnibus normality test was performed on all data to test for normality. Cell harvest data were nonparametric data and were analyzed with a Wilcoxon matchedpairs signed rank test, and data reported as median and range. Significance was declared at P < 0.05. Data were analyzed with a statistical software program Graph Pad Prism, San Diego, CA. The effect of tensioning meniscal fibrochondrocytes on ECM composition was analyzed using a two-tailed Student’s t-test. The effect of different cell types (synoviocytes as TSB and meniscofibrochondrocytes as TMB) on gene expression, ECM synthesis, and viability was analyzed using a paired two-tailed Student’s t-test. Significance was declared at P < 0.05. These data were analyzed using Statistical Analysis System, version 9.2 (SAS Institute Inc.).

Table 1 The effect of tissue source on fibrochondrogenic gene expression of tensioned neotissues (fold changesa ± SEM). Tensioned neotissues (TN) Tissue source Gene: SOX-9 Collagen type I α1 Collagen type II α1 Aggrecan Interleukin-6 Tumor necrosis factor α

Synovium N=6

Meniscofibrochondrocytes N=6

SEM

P-value

0Reference 0 0 0 0 0

+1.21 +2.01 −12.6 −2.02 −2.11 +1.11

0.51 1.14 1.89 0.39 1.86 0.89

0.64 0.40 0.03 0.05 0.49 0.85

a Fold changes were calculated using the following formula: fold change = 2 − ΔΔ CT = [(C T gene of interest − C T housekeeping gene GAPDH) TMB − (C T gene of interest − CThousekeeping gene GAPDH)TSB].

Please cite this article in press as: George A. Ballard, et al., Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog, Research in Veterinary Science (2014), doi: 10.1016/j.rvsc.2014.05.002

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unraveling or slipping off the wire hoops. During long term culture (≥28 days), an average of 3.4 TMB and TSB (range 2–5 bioscaffolds) per dog slipped off their wire hoops, resulting in contracted masses, and were not analyzed in the present study. With wires removed, the mean dry tissue weight of TMB was 27.1 mg ± 7, which was not different from 23.4 mg ± 4 for TSB (P = 0.64). 3.3. Gene expression

Fig. 1. Representative samples of a tensioned meniscal fibrochondrocyte bioscaffold, or TMB (‘M’) and a tensioned synoviocyte bioscaffold, or TSB (‘S’).

3. Results

Gene expression of type II collagen (P = 0.03) and aggrecan (P = 0.05) was lower in TMB as compared to TSB, whereas no significant differences were observed for relative expression of type I collagen (P = 0.40), SOX-9 (P = 0.64), TNF-α (P = 0.85), and IL-6 (P = 0.49; Table 1). Gene expression of IL1β was below the detection limit. For comparison, we also determined the relative gene expression of one meniscal CS, which had a 35-fold lower gene expression of type II collagen and aggrecan than the TMB sheet of the corresponding dog.

3.1. Cell harvest from arthroscopic debris 3.4. Glycosaminoglycan content The mean age of dogs presented for treatment of meniscal tears was 5.7 years (range 2–8 years); there were three male neutered dogs, two female spayed dogs, and one female intact dog. Breeds represented included two Australian Shepherds, one Golden Retriever, one Rottweiler, one American Staffordshire Terrier, and one mixed breed. All dogs had synovitis, osteophytosis of the terminal sulci, and grade 1–2 Outerbridge (Outerbridge, 1961) lesions of the medial tibial plateau and medial femoral condyle. A median of 1.58 g of synovium (0.76–2.7 g, wet weight) and a median of 0.21 g of meniscus (range, 0.09–0.60 g wet weight) was removed per dog (P = 0.04). The majority of cells recovered from synovial tissue were red blood cells; median nucleated cell count yield was 3.68 × 105 (1.30 × 105–1.68 × 106 cells) for synovium and 3.05 × 105 (1.05 × 105–1.46 × 106 cells) for meniscectomized tissue (P = 0.71); nucleated cell count standardized to wet weight was also not significant between tissue types (P = 0.81). All cells obtained after tissue digest were 100% viable according to the trypan blue exclusion assay. 3.2. Tensioned bioscaffold culture Median time from cell harvest to TMB and TSB formation was similar between cell types, at 37 days (range 28–52 days). The appearance of representative TMB and TSB are pictured in Fig. 1. By days 7–8 in culture TMB and TSB were stable to movement without

There was no difference in total GAG content of meniscal CS (591 µg ± 185) and TMB (P = 0.55), nor TMB (454 µg ± 127) versus TSB (426 µ g ± 86; P = 0.88). A greater proportion of TMB was GAG (1.7% ± 0.10) versus meniscal fibrochondrocyte CS (0.9% ± 0.41, P = 0.01), while there was no difference in proportion of GAG content of TMB versus TSB (2.0% ± 0.40, P = 0.48). After adjusting to a normal distribution by natural logarithmic transformation, the chondrogenic index (a measure of cellular GAG production, with GAG standardized to dsDNA content) was higher in TMB versus meniscal fibrochondrocyte CS (P = 0.04), but not in TMB versus TSB (P = 0.40, Table 2). 3.5. Collagen content There was no difference in total collagen content of meniscal CS (2812 µg ± 354) and TMB (P = 0.41), nor TMB (4109 µg ± 1077) and TSB (3963 µg ± 731 P = 0.92). In contrast, TMB had a higher collagen concentration (17.0% ± 2.3) than meniscal fibrochondrocyte CS (5.06% ± 0.48, P = 0.006), however, there was no difference in percent collagen content of TMB versus TSB (18.3% ± 3.7, P = 0.51). Collagen index, a measure of cellular collagen production, was not different between meniscal fibrochondrocyte CS and TMB (P = 0.12), and TSB versus TMB (P = 0.46, Table 2).

Table 2 The effect of tissue source on extracellular matrix content (mean ± SEM). Contrastsa

Tissue source Construct type Tension/Tissue source

Concentrations (µg/bioscaffold): GAG Collagen DNA Proportion (% dry weight): GAG Collagen DNA Index (μg/µg dsDNA): GAG Collagen

CS

TMB

TSB

Presheet/Meniscus

Present/Meniscus

Present/Synoviocytes

N=3

N=5

N=5

Tensioned

Meniscus

591 ± 185 2812 ± 354 126 ± 51

454 ± 127 4109 ± 1077 52.4 ± 14.9

426 ± 86 3963 ± 731 30.0 ± 4.5

0.55 0.41 0.28

0.88 0.92 0.29

0.99 ± 0.41 5.06 ± 0.48 0.22 ± 0.07

1.77 ± 0.10 17.0 ± 2.3 0.23 ± 0.05

2.03 ± 0.40 18.3 ± 3.7 0.16 ± 0.06

0.01 0.006 0.91

0.48 0.51 0.46

7.12 ± 4.43 29.2 ± 10.2

10.2 ± 2.7 98.4 ± 28.1

14.9 ± 3.1 140 ± 31

0.55 0.12

0.40 0.46

a P-values for statistical contrasts. ‘Tensioned’ compares meniscal tissue that was (TMB) or was not on dreamcatcher (CS), using a Student’s t-test. ‘Meniscus’ compares meniscal (TMB) and synovial tissue (TSB) from the same dog, using a paired t-test.

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3.6. Cell viability and cellularity Cell viability: Fluorescent microscopy revealed layers of fibroblastic cells in TSB, oriented in parallel. Cells in TMB had both fibroblastic and rounded phenotypes. Due to the complex threedimensional structure of TMB and TSB viability cell counts represented an estimation. Cell viability was estimated to be 60– 70% for TSB and 30–40% for TMB. Cell mortality did not appear to correlate with a peripheral versus central location on the bioscaffolds. Percent dsDNA content was used to quantify tissue cellularity, and was not significantly different between meniscal fibrochondrocyte CS (126 µg ± 51, or 0.22% ± 0.07 of dry weight) and TMB (52.4 µg ± 14.9, or 0.23% ± 0.05 of dry weight P = 0.28; P = 0.91), nor between TMB and TSB (30.0 µg ± 4.5, or 0.16% ± 0.06 of dry weight; P = 0.29; P = 0.46, Table 2). 3.7. Histological analysis H&E staining showed that TSB cells appeared fibroblastic, while TMB contained both fibroblastic type cells and round cells located in pseudo-lacunae; TMB and TSB were highly cellular. Both bioscaffold types had heterogenous eosinophilic ECM. Regions of cellular disintegration, karyolysis and amorphous debris consistent with cell death were observed in TMB (Fig. 2). The majority of cells in TSB were moderately to strongly immunoreactive to ASM, while 25% of cells in TMB were moderately immunoreactive and appeared to be located around the periphery of the bioscaffold. Additionally TMB contained multiple holes, ranging

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from 50–300 µm, which were ringed with cells strongly immunoreactive to ASM (Fig. 2). Trichrome staining revealed that TMB contained dense collagen deposited in whorls and patches; this tissue appeared more disorganized. In contrast, TSB contained regions of collagen oriented in bands and sheets parallel to the vector of tension (Fig. 3). Widespread strong extracellular immunoreactivity to type I collagen was present in TMB, while TSB contained moderate extracellular collagen I immunoreactivity; both bioscaffold types contained >50% of cells with moderate to strong intracellular immunoreactivity. Regional mild to moderate extracellular immunoreactivity to type 2 collagen was noted in all TMB, while regional mild extracellular immunoreactivity was seen in 3/6 TSB. Intracellular immunoreactivity to type II collagen was present in >50% of cells in TMB and in 10–50% of cells in TSB. Toluidine blue staining revealed regional GAG deposition in all constructs, which were more dense in TMB versus TSB (Fig. 4). 4. Discussion In this study we found that meniscal and synovial arthroscopic debris obtained from dogs undergoing treatment for naturally occurring meniscal injury can be used to obtain viable cells for culture. Contrary to our working hypothesis, cell viability of meniscal debris was high. Our meniscal fibrochondrocytes originated from nondisplaced bucket handle tears, which permit generation of some hoops strains (Jones et al., 1996), and thus these cells may have had a higher viability than more unstable and diseased tear types, such

Fig. 2. Hematoxylin and Eosin stain of meniscal fibrochondrocyte bioscaffolds (‘MH&E’) and synoviocyte bioscaffolds (‘SH&E’). Note the fibroblastic cells and longitudinal orientation of TSB ECM. Arrows indicate regions of cellular disintegration, karyolysis and amorphous debris consistent with cell death in TMB. Immunohistochemisty for ASM and negative controls of meniscal fibrochondrocyte bioscaffolds (‘MNC’ and ‘MASM’) and synoviocyte bioscaffolds (‘SNC’ and ‘SASM’). Note the global moderate expression of ASM in the synoviocytes, versus regional strong ASM expression along the periphery of spontaneously forming circular defects in the meniscal fibrochondrocytes. 10× objective magnification, bar = 100 μm.

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Fig. 3. Masson’s Trichrome staining for collagen of meniscal fibrochondrocyte bioscaffolds (‘MMT’) and synoviocyte bisocaffolds (‘SMT’). Immunohistochemistry for type I collagen and type II collagen of meniscal fibrochondrocyte bioscaffolds (‘Mcol1’ and ‘Mcol2’) and synoviocyte bioscaffolds (‘Scol1’ and ‘Scol2’). Arrows indicate rounded cells in lacunae. Note the longitudinal organization of collagen in the synoviocyte bisocaffolds, and the greater immunoreactivity to type II collagen in the meniscal fibrochondrocyte bioscaffolds. 10× objective magnification, bar = 100 μm.

as flap tears, displaced bucket handle tears, or macerated complex tears. In addition, despite the harvest of two very different tissue types and a greater harvested synovial wet weight, nucleated cell yield from synovial and meniscal arthroscopic debris was similar, indicating a blood dilution effect for synovial tissues. We also determined that meniscal fibrochondrocytes cultured as TMB increased the proportion of GAG and collagen relative to CS hyperconfluent monolayer culture. This is similar to previous find-

Fig. 4. Toluidine Blue staining for glycosaminoglycan of meniscal fibrochondrocyte bioscaffolds (‘MTB’) and synoviocyte bisocaffolds (‘STB’). Note the longitudinal organization of ECM in the synoviocyte bisocaffolds. 10× objective magnification, bar = 100 μm.

ings of canine synoviocyte hyperconfluent monolayer, culture versus TSB (Warnock et al., 2013). The longer duration of culture may have increased proportions of GAG and collagen, which has been found in human meniscal fibrochondrocytes (Baker et al., 2009). Meniscal fibrocartilages typically experience tensile load (Messner and Gao, 1998); conceivably, application of tensile load as applied in this study also increased GAG and collagen proportions in TMB, versus CS (Messner and Gao, 1998). As tensioned bioscaffolds, both synoviocytes and meniscal fibrochondrocytes produced the collagens and GAG found in the normal meniscus. Histologically, TMB contained more components of the axial meniscus, including rounded cells in lacunae and type II collagen (Fig. 4). This is consistent with the axial origin of the meniscectomized debris and harvested meniscal fibrochondrocytes used in this study (Hellio Le Graverand et al., 2001a, 2001b; Kambic and McDevitt, 2005). In comparison, TMB collagen (17.0%) and GAG concentrations (1.77%) were lower than the 60–70% collagen and 2–3% GAG per dry weight of the whole meniscus (McDevitt and Webber, 1990; Stephan et al., 1998). It is interesting to note that in vitro human meniscal fibrochondrocyte constructs also contained lower collagen (11.5%) and GAG (2%) (Baker et al., 2009) than native meniscal tissue, and were comparable to the TMB of our study. A number of factors contributed to the lower collagen and GAG content of TMB relative to native meniscal tissue; the biological and biomechanical in vitro environment of the present study may not have allowed the meniscal fibrochondrocytes to achieve their full synthetic potential. All meniscal tissues in this study originated from the avascular axial rim of the meniscus, which experiences more compressive forces versus the abaxial rim (Fithian et al., 1990;

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Sweigart et al., 2004). Additionally, the primary biomechanical stimulus for GAG and collagen type II formation is compression (Mauck et al., 2003). Thus axial meniscal cells may require addition of in vitro compression to induce more collagen and GAG formation (Huey and Athanasiou, 2011; Puetzer et al., 2012), versus exposure to the mild tensile forces applied in TMB culture. In support of this theory, TMB fibrochondrocytes had low expression of type II collagen and aggrecan genes. Axial meniscal cells are very sensitive to oxygen tension; when hypoxic, they increase production of types I and II collagen (Adesida et al., 2007). Unfortunately, cells in the present study were exposed to atmospheric oxygen concentrations. Thus, different culture conditions should be applied in future studies utilizing meniscal fibrochondrocytes. The origin of the cells used in this study may also account for lack of TMB and TSB matrix quantities. Axial meniscal fibrochondrocytes have decreased differentiation (Mauck et al., 2007), healing capacity (Kobayashi et al., 2004), and ability to produce collagen post wounding (Spindler et al., 1994) as compared to meniscal fibrochondrocytes of the abaxial meniscus. Additionally, both synoviocytes and meniscal fibrochondrocytes originated from diseased tissue, which possibly decreased ECM formation. In contrast to TMB, TSB contained more collagen (18.3%) and GAG (2.03%) than the 11% collagen and 0.7% GAG of normal, native synovium (Price et al., 1996). This may be due to a change in the biomechanical environment, from the mobile, non-weight bearing of native synovium to the in vitro environment of TSB. The making of TSB requires application of tensile forces, and synovial ASM expression results in self-tensioning (Vickers et al., 2004) of TSB (Warnock et al., 2013). Synoviocytes respond to tensile forces by increasing collagen (Warnock et al., 2013) and hyaluronic acid synthesis (Momberger et al., 2005; Sakamoto et al., 2010). As compared to the disorganized histologic architecture of TMB, TSB responded to tension with collagen arranged into regions of parallel sheets and bands. Forming organized histologic structure is an important step toward recreating the arrayed collagen structure of the meniscus in vitro. The meniscal bioscaffolds had lower cell viability compared to TSB. In native tissue, meniscal fibrochondroctyes achieve homeostasis at 2.65–4.3% tensile strain (Jones et al., 1996). It is possible that during removal and handling of the hyperconfluent cell sheet to make TMB, strain briefly exceeded 4.3%. Concomitantly, culture in monolayer and culture as TMB could have exposed to fibrochondocytes to very low strain, similar to the low tensile strain of torn meniscal tissue (Jones et al., 1996). Excessive or deficient force can result in cell apoptosis and induce an inflammatory state in rat tenocytes (Arnoczky et al., 2008; Egerbacher et al., 2008; Ferretti et al., 2006); it is plausible to apply these principles to meniscal fibrochondrocytes. Evidence for inappropriate biomechanical stimulation can be found in the histologic ECM disorganization of TMB with regions of high cell mortality. Low cell viability in TMB also had the additive effect of decreased in vitro ECM production. Thus, application of static tension, as used in the present study, likely negatively impacted the health and viability of the meniscal fibrochondrocytes, and is not recommended. Bioscaffold cellularity, as measured by the dsDNA assay, was also not different between TMB and TSB, despite marked viability differences. The dense ECM framework around TMB cells may have prevented dead cell debris from being washed away during media changes. Bioscaffold cellularity was likely established at the formation of the hyperconfluent monolayer cell sheets, as seen when comparing dsDNA content of CS to TMB, which resulted in similar cell concentrations due to cellular contact inhibition (Stoker and Rubin, 1967). Meniscal primordia are highly cellular structures (Clark and Ogden, 1983), similar to the bioscaffolds in this study. Future culture techniques will need to decrease cellularity and increase ECM to approximate the adult native meniscus, which has a sparse cellular population (Clark and Ogden, 1983).

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There was widespread of expression of ASM in TSB versus more intermittent ASM expression in TMB. Strong expression of ASM has been found previously in canine synovium (Kambic et al., 2000; Vickers et al., 2004), and is involved in self-tensioning of TSB (Warnock et al., 2013). Approximately 10–25% of meniscal fibrochondrocytes express α -smooth muscle actin, conferring microcontractile properties to the cells (Ahluwalia et al., 2001; Kambic et al., 2000; Spector, 2001), which is thought to be involved with meniscal reparative responses (Kambic et al., 2000). In contrast, meniscal fibrochondrocyte ASM expression in tensioned bioscaffolds resulted in formation of spontaneous holes, thereby playing a destructive role. The lack of cell orientation and ASM mediated self-tensioning of meniscal fibrochondrocytes may have additionally lead to a biomechanically unfavorable environment in TMB, poor ECM formation and organization, and cell mortality. 5. Conclusion Axial meniscal debris in dogs obtained during arthroscopic partial meniscectomy is a viable source of meniscal fibrochondrocytes for tissue engineering purposes. We accept the null hypothesis regarding meniscal fibrochondrocyte versus synoviocyte bioscaffold ECM quantity, but reject the null hypothesis regarding cell viability, ASM expression, and type II collagen content of TMB versus TSB. The confirmation of production of specific meniscal-ECM components gives promise to either synoviocytes or meniscal fibrochondrocytes as a viable cell source for meniscal tissue engineering. Additionally, organization of collagen in TSB shows promise for developing the structure needed to create a meniscal implant. Despite low cell viability, TMB meniscal fibrochondrocytes were able to synthesize dense collagen and GAG; improving cell viability may dramatically increase the ECM content. Thus, autologous, diseased meniscal fibrochondrocytes and synoviocytes are a possible cell source for future meniscal tissue engineering research, with the goal of producing a meniscal implant, however culture of meniscal fibrochondrocytes as TMB may not be an ideal method to maximize ECM formation potential. Acknowledgments This study was funded by Oregon State University, College of Veterinary Medicine, and the Morris Animal Foundation. Presented in part as an abstract at the Veterinary Orthopedic Society Meeting, Crested Butte, Colorado, U.S.A., 2012. Appendix Assays used for RT-PCR Gene

Amplicon size

Assay catalog number

Reference sequence

Interleukin 1, beta Interleukin 6 Tumor necrosis factor SOX-9 Collagen, type I, alpha 1 Collagen, type II, alpha 1 Aggrecan GAPDH

70 68 131 103 87 89 125 97

Cf02671952_m1 Cf02624151_m1 Cf02628237_m1 Cf02625134_g1 Cf02623126_m1 Cf02622862_m1 Cf02674826_m1 AIWRF9W

NM_001037971.1 NM_001003301.1 NM_001003244.4 NM_001002978.1 NM_001003090.1 NM_001006951.1 NM_001113455.1 NM_001003142.1

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