Korean Journal of
Veterinary Service

Osteoarthritis (OA) is defined as the progressive loss of articular cartilage, associated with changes in periarticular osteophytosis, subchondral bone metabolism, and a variable degree of inflammation in the synovial membrane, fat pad, and meniscus (Man and Mologhianu, 2014; Ripmeester et al., 2018; Zheng et al., 2021). OA is common in small animals and is known to affect 20% of adult dogs (increasing to 80% in dogs aged 8 years and older) and 60% of adult cats (Johnston, 1997; Hardie et al., 2002). Articular cartilage consists of an extracellular matrix (ECM), which includes a highly organized network of collagens, proteoglycans, and hyaluronan. These collagens provide the tissue with tensile strength and shape and are primarily composed of collagen type II (COL2A1) fibrils, which make up to 90% of the total collagen content (Thielen et al., 2019). The ECM plays a crucial role in forming the supportive skeleton of cartilage and maintaining its shape.

OA is influenced by the expression of several inflammatory cytokines and genes. Articular chondrocytes play a crucial role in maintaining cartilage homeostasis by balancing catabolic and anabolic effects (Kim et al., 2015). OA leads to the degradation of essential cartilage matrix components, such as COL2A1 and aggrecan, due to the increased production of proteolytic enzymes (Hunter, 2011). The inflammatory cytokines involved in the catabolic changes in OA include interleukin (IL)-1β and tumor necrosis factor-α (TNF)-α (Thielen et al., 2019). Studies have shown that during OA progression, a switch occurs from COL2A1 to collagen type I (COL1A1) (Miosge et al., 2004).

OA can be classified as idiopathic or secondary. In dogs, the disorder most frequently occurs secondary to an identifiable joint abnormality, such as a developmental disorder, joint instability, or trauma (e.g., osteochondritis dissecans, hip dysplasia, or cruciate ligament rupture) (Ku et al., 2009). The management of osteoarthritis is typically conservative and multimodal. Most cases are treated with nonsteroidal anti-inflammatory drugs (NSAIDs) combined with nutritional, weight, and exercise management. However, long-term use of NSAIDs can cause adverse effects on the gastrointestinal, cardiovascular, and renal systems (Harirforoosh et al., 2013). Prolonged NSAID treatment directly reduces COX-2 and PGE-2 production in articular cartilage in osteoarthritis patients, which adversely affects cartilage repair (Alvarez-Soria et al., 2008).

Meanwhile, cytokines that cause anabolic changes include over 30 members of the transforming growth factor β (TGF-β) family, such as the TGF-βs, activins, bone morphogenetic proteins (BMPs), and growth/differentiation factors (GDFs). Multiple BMPs, including BMP-2, BMP-7, BMP-4, BMP-6, BMP-9, and BMP-14, play important roles in chondrocyte biology (Chen et al., 2004; van Caam et al., 2016). Among these, BMP-2 is known to induce chondrocyte formation, osteoblast differentiation, and bone healing. In both healthy and OA cartilage, BMP-2 mRNA expression can be detected (Pregizer and Mortlock, 2015). BMP-2 signaling promotes ECM production and proliferation, increases proteoglycan content, and enhances COL2A1 expression (Hills et al., 2005).

Pro-inflammatory cytokines such as IL-1β can induce BMP-2 expression in chondrocytes (Fukui et al., 2003). In early OA, elevated BMP-2 levels in damaged cartilage may increase ECM synthesis, promoting tissue repair while also inducing cartilage degradation by stimulating matrix metalloproteinase (MMP)-13 expression (Nakase et al., 2003; Papathanasiou et al., 2012). This response fails to repair cartilage due to the predominance of catabolic activity (Van der Kraan et al., 2010). As OA progresses, chondrocytes break down and adopt a hypertrophic-like phenotype (Papathanasiou et al., 2012; Singh et al., 2019). Additionally, one report suggests that BMP-2 does not affect chondrocytes directly but instead contributes to osteophyte formation around cartilage (Blaney Davidson et al. 2015).

Platelet-rich plasma (PRP) is an emerging material in regenerative medicine and contains various growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β (Arnoczky et al., 2011). PRP has been shown to increase the proliferation of chondrocytes, reduce apoptosis, and enhance autophagy in human osteoarthritic chondrocytes (Moussa et al., 2017). Additionally, PRP is known to possess anti-inflammatory properties (Zhang et al., 2013).

The effects of PRP or BMP-2 alone on chondrocyte differentiation, ECM maturation, and synthesis have been investigated in many studies in human medicine (Gründer et al., 2004; Asjid et al., 2019). However, no studies have examined the combination of PRP and BMP-2 or their effects on canine chondrocytes. Therefore, the purpose of this study is to determine the effects of PRP and BMP-2 on proliferation, inflammation, and ECM-related gene expression in canine inflammation-induced chondrocytes.

Preparation of activated platelet-rich plasma (PRP)

To prepare the PRP, acid citrate dextrose-A solution (Northrom solution, Korea) and whole blood from a 5-year-old healthy beagle were mixed at a ratio of 1:7. Radiographic examination of the stifle, CBC, serum chemistry, and synovial fluid analysis were performed to confirm the beagle’s health. PRP was collected using a double-centrifuge protocol. After centrifugation at 2,500 rpm for 10 minutes, the upper 70% of the platelet-poor plasma was discarded, and the remaining plasma and buffy coat were collected through a second centrifugation at 2,500 rpm for 5 minutes. Then, 10% CaCl₂ was added to activate the platelets. The produced material was centrifuged again, and the supernatant was used. PRP was defined as having a platelet concentration greater than 1.0×10⁶ platelets/µL.

Isolation of canine chondrocyte

Articular cartilage from the stifle joint was collected aseptically from another beagle (4.5 year old, 6 kg). The dog was considers as healthy, according to physical examinations, CBC and serum chemistry result. The dog was euthanized using T-61. The joint showed no gross abnormalities, such as degeneration, bone remodeling, or joint swelling. Samples were collected from a depth of 2 mm or less in the articular cartilage to avoid including subchondral osteocytes. These procedures were approved by the Kyungpook Animal Ethics Committee (No. KNU 2021-0171). The harvested cartilage was washed four times with PBS, minced to 1 mm³ or less, and then centrifuged at room temperature to remove debris. The cartilage was treated with 0.8 U/mg dispase II (Sigma-Aldrich, USA) in Dulbecco’s Modified Eagle Medium (DMEM)-low glucose (LG) (Sigma-Aldrich, USA) containing 100 μg/mL penicillin and 100 μg/mL streptomycin (PSA, Sigma-Aldrich, USA). After pre-digestion, the sample was centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded. The remaining material was digested with 0.2% collagenase (Sigma-Aldrich, USA) for 10 hours and then centrifuged at 1,000 rpm for 5 minutes. The remaining debris was removed through a 100-μm nylon mesh. The harvested chondrocytes were seeded in 100×20 mm cell culture dishes. After 24 hours, non-adherent cells were removed by washing with PBS, and the medium containing DMEM-LG, 10% fetal bovine serum (FBS) (Gibco, USA), 1% penicillin, and 1% streptomycin (Gibco, USA) was replaced every 72 hours. Chondrocytes were used at passage 3 for all experiments.

LPS-induced inflammation

After isolation, passage 3 chondrocytes were starved by replacing the medium with serum-free medium for 24 hours to induce inflammation. After that, inflammation was induced by treating the cells with 10 μg/mL of lipopolysaccharide (LPS) for 6 hours.

Proliferation of chondrocytes in monolayer cultures

Chondrocytes (1.25×10⁴ cells/well) were seeded in a 96-well plate with DMEM-LG, 10% FBS, and 1% penicillin-streptomycin (PS) for 24 hours. The floating cells were discarded, and the medium was changed to induce inflammation in the same manner as described in above section. After removal of LPS, each group mentioned below was treated for 24 hours including control group (DMEM-LG/1% PS with 10 μg/mL LPS), BMP group (DMEM-LG/1% PS with 10 μg/mL LPS+200 ng/mL BMP-2 (CGBIO, Korea)), PRP group (DMEM-LG/1% PS with 10 μg/mL LPS+5% PRP), and PRP+BMP group (DMEM-LG/1% PS with 10 μg/mL LPS+5% PRP+200 ng/mL BMP-2). After 24 hours of treatment, Cell Count Kit (CCK)-8 (Dojindo Molecular Technologies, Inc., Japan) was added (10 μL/well) to assess cell proliferation. Using a microplate spectrophotometer (Epoch, Biotek Instruments, USA), absorbance was measured at 450 nm after 1 hour.

Gene expression analysis (real-time q-PCR)

Chondrocytes were cultured in 100×20 mm cell culture dishes containing DMEM-LG, 10% FBS, and 1% penicillin and streptomycin. Once confluence was reached, starvation, inflammation, and treatment were performed as described above. Real-time qPCR was performed using a Bio-Rad CFX96 (Bio-Rad Laboratories, Inc., USA). RNA was extracted using TRIzol^® Reagent (Life Technologies, USA). According to the manufacturer’s protocol, cDNA was synthesized using M-MuLV reverse transcriptase (Cosmogenetech, Seoul, Korea) and RNase Inhibitor (Cosmogenetech, Seoul, Korea). Primers for COL1A1, COL2A1, IL-1β, and Osteocalcin were calculated. GAPDH was used as the housekeeping gene (Table 1).

Table 1 . Sequences of PCR primers.

Gene	Primer	Sequence (5’-3’)
GAPDH	Forward Reverse	GTT TGT GAT GGG CGT GAA CC TTT GGC TAG AGG AGC CAA GC
COL1A1	Forward Reverse	CAT CCA GCT GAC CTT CCT GC CTC CAG TGT GAC TCG TGC AG
COL2A1	Forward Reverse	GTG GAC GTT CAG GCG AAA CT CTC GGC ATC ATG CTG TCT CAG
IL-1β	Forward Reverse	TGA GGC ATT TCG TGT CAG TCA TCC TGT AAC TTG CAG TCC ACC
Osteocalcin	Forward Reverse	AGC AGC AGC TGC TCA CAG A CTT GGA CAC GAA GGC TGC AC

Statistical analysis

All data were recorded as the mean±standard deviation (SD). Based on Levene’s test results, the Mann-Whitney U test or ANOVA followed by Scheffe’s post-hoc test was performed to compare groups (SPSS Statistics version 23, IBM SPSS Inc., USA). P-values less than 0.05 were considered statistically significant.

Proliferation test

Compared to the control group, the BMP group did not show a significant increase (P=0.442). Cell proliferation was increased in the PRP groups compared with the control and BMP groups (P<0.05, P=0.002). However, there was no significant difference between the PRP group and the PRP+BMP group (P=0.505) (Fig. 1, 2).

Figure 1. Microscopic feature of canine chondrocytes after 24 hours treatment. All groups were treated (A) Control, (B) 200 ng/mL BMP-2, (C) 5% PRP and (D) 200 ng/mL BMP-2 with 5% PRP for 24 hours after 10 μg/mL LPS-induced inflammation.

Figure 2. Result of proliferation test of canine chondrocytes after 24 hours treatment. All groups were treated on DMEM with no serum, 200 ng/mL BMP-2, 5% PRP, 200 ng/mL BMP-2 with PRP, respectively for 24 hours after 10 μg/mL LPS-induced inflammation. Data were expressed as mean±SD.
*Significant differences compared with control group (P<0.05).
^#Significant differences compared with BMP group (P<0.05).

Real-time PCR analysis

COL1A1 and COL2A1 were significantly increased in the BMP group compared to the control group (P<0.05). On the other hand, the PRP group and PRP+BMP group showed a significant decrease compared with the control group (P<0.05) (Fig. 3, 4). IL-1β was significantly decreased in the PRP and PRP+BMP groups (Fig. 5). Osteocalcin showed no significant difference between the control group and BMP group and was significantly decreased in the PRP and PRP+BMP groups (Fig. 6).

Figure 3. Relative mRNA expression of COL1A1 on canine chondrocytes in LPS-induced inflammatory conditions after 24 hours treatment by Real-time PCR on control, 200 ng/mL BMP-2, 5% PRP, and combination of 5% PRP and 200 ng/mL BMP-2, respectively. Data were expressed as mean±SD.
*Significant differences compared with control group (P<0.05).
^#Significant differences compared with BMP group (P<0.05).

Figure 4. Relative mRNA expression of COL2A1 on canine chondrocytes in LPS-induced inflammatory conditions after 24 hours treatment by Real-time PCR on control, 200 ng/mL BMP-2, 5% PRP, and combination of 5% PRP and 200 ng/mL BMP-2, respectively. Data were expressed as mean±SD.
*Significant differences compared with control group (P<0.05).
^#Significant differences compared with BMP group (P<0.05).

Figure 5. Relative mRNA expression of IL-1β on canine chondrocytes in LPS-induced inflammatory conditions after 24 hours treatment by Real-time PCR on control, 200 ng/mL BMP-2, 5% PRP, and combination of 5% PRP and 200 ng/mL BMP-2, respectively. Data were expressed as mean±SD.
*Significant differences compared with control group (P<0.05).
^#Significant differences compared with BMP group (P<0.05).

Figure 6. Relative mRNA expression of Osteocalcin on canine chondrocytes in LPS-induced inflammatory conditions after 24 hours treatment by Real-time PCR on control, 200 ng/mL BMP-2, 5% PRP, and combination of 5% PRP and 200 ng/mL BMP-2, respectively. Data were expressed as mean±SD.
*Significant differences compared with control group (P<0.05).
^#Significant differences compared with BMP group (P<0.05).

In this study, we examined the proliferation and gene expression of chondrocytes in the presence or absence of PRP and BMP-2 under LPS-induced inflammatory conditions. These results can serve as basic data for regenerative medicine treatments for OA. OA refers to a condition in which the balance between inflammatory cytokines exhibiting catabolic activity and TGF-β family members exhibiting anabolic activity is disrupted. The OA process is associated with pro-inflammatory cytokines such as IL-1β and TNF-α (Kim et al., 2019). Most of the growth factors found in PRP have been shown to enhance the proliferation of articular chondrocytes (Akeda et al., 2006). Additionally, the anti-inflammatory action of platelet-rich plasma on human chondrocytes through NF-κB inhibition is well established (Bendinelli et al., 2010). In our study, PRP was effective in promoting proliferation and exerting anti-inflammatory effects on chondrocytes under inflammatory conditions. However, BMP-2 did not show a significant increase in the proliferation test. The lack of effect of BMP-2 on proliferation in our study may be due to the short treatment time of 24 hours.

This articular cartilage is composed of chondrocytes and an extracellular matrix. The extracellular matrix, or ECM for short, is made up of collagen type II, which accounts for more than 90% of the ECM, as well as other collagens, proteoglycans, hyaluronan, and more. These ECM plays an essential role in forming and maintaining the supporting structure of cartilage (Thielen et al., 2019). The progression of OA alters the composition ratio of COL2A1 and COL1A1, leading to the degradation of the ECM (Miosge et al., 2004). When OA occurs, inflammatory cytokines promote BMP-2 signaling. It has been reported that BMP-2 signaling induces ECM production and cell proliferation (Hills et al., 2005). Thus, BMP-2 could play an important role in cartilage repair and chondrogenesis (Medvedeva et al., 2018; Salazar et al., 2016). Consistent with previous studies, BMP-2 could elevate COL2A1. However, when PRP and BMP-2 were combined, COL2A1 gene expression decreased.

BMP-2 is known to have both osteogenic and cartilage differentiation properties. Osteocalcin is a marker of osteoblast differentiation of stem cells and can also be found in post-hypertrophic chondrocytes. It has been found that oteocalcin mRNA expression increases as OA progresses gradually (Pullig et al., 2000). Interestingly, in this study, BMP-2 did not increase osteocalcin gene expression, which is associated with the differentiation of osteoblast. The expression of oteocalcin was decreased in both groups treated with PRP. This suggests the potential of PRP to inhibit the osteogenic capacity of chondrocytes. The decrease in osteocalcin expression in the PRP group may be due to the anti-inflammatory action of PRP.

There are also limitations to this study. First, this study was conducted using a monolayer model to collect basic data. In monolayer cultures of chondrocytes, the phenotype could change as the passage progresses (Ma et al., 2013). Therefore, the level of gene expression may differ. Cultures in 3D systems, such as alginate beads, could be more effective for maintaining the phenotype of chondrocytes than monolayer cultures (De Ceuninck et al., 2004). Therefore, experiments with 3D-cultured cells or in vivo studies are necessary to further validate these findings. Additionally, in human medicine, the more severe the OA, the higher the osteocalcin expression level (Pullig et al., 2000). In one study examining the short-term expression of BMP-2, BMP-2 secretion during the first 4 days was not sufficient to induce MSC differentiation, whereas 6-day expression induced the formation of hypertrophic cartilage and bone marrow-filled bone (Noël et al., 2004). Therefore, the unchanged level of osteocalcin expression could be due to the short duration of BMP-2 treatment. Further research is recommended to investigate the long-term effects of BMP-2 treatment, with or without PRP, on chondrocytes in an inflammatory state to confirm the presence of hypertrophic chondrocytes or osteoblast cells.

Our results show that PRP promoted the proliferation of canine chondrocytes under inflammatory conditions, exhibited anti-inflammatory effects, and significantly decreased osteocalcin expression. Therefore, it is possible that PRP may inhibit the differentiation of chondrocytes into hypertrophic chondrocytes, as well as inhibit osteoblast differentiation. Consistent with previous studies, BMP-2 upregulated the COL2A1 gene expression, which is a component of the ECM. However, BMP-2 had no effect on proliferation or inflammation. Furthermore, PRP blocked BMP-2 from elevating COL2A1 gene expression.

No potential conflict of interest relevant to this article was reported.