In the current study, we extracted hPRP from human blood and investigated the combined treatment of hPRP and HA on the recovery of TMJ-OA from clinical trials in dish to humans. First, for clinical trial in dish, chondrocytes isolated from rat TMJ were used as an in-vitro (2D and 3D)-TMJ-OA model which was created by pro-inflammatory cytokines, namely IL-1β + TNF-α, designated as I + T. Moreover, a 3D TMJ-OA model was also established, in which a condyle-like structure was formed using collagen scaffold–encapsulated culture under the I + T condition. Second, for pre-clinical trials in animals, an in-vivo TMJ-OA rat model was created by CFA administration to evaluate condylar degeneration. Finally, we recruited patients with TMJ OA for the human trial. Thus, the effects of combine therapy of hPRP/HA on TMJ-OA recovery was intensively examined as summarized in Fig. 1.

Fig. 1

Schematic of hPRP/HA treatment in TMJ-OA disease from bench to beside

Effect of hPRP/HA therapy on proliferation of chondrocytes in TMJ-OA in-vitro 2D mode

To evaluate the optimal HA and hPRP concentrations on rat TMJ chondrocytes, isolated chondrocytes from TMJ were treated with increasing concentrations of HA or hPRP. The dose for most viability of rat TMJ chondrocytes was 250 μg/mL for HA and 1 ng/mL for hPRP (Fig. 2A, B). Thus, MTT assay results indicated that 250 μg/mL HA and 1 ng/mL hPRP were deemed as their optimal concentrations for further tests of the hPRP/HA therapy. A cellular TMJ-OA 2D model was constructed with IL-1β + TNF-α (I + T) to induce inflammation in rat TMJ chondrocytes to mimic the in-vivo system. The chondrocytes were cultured in the (I + T)-conditioned medium and then treated with or without hPRP/HA for 48 h. Under the I + T condition, the cell number showed a significant decrease, and fibroblast-like morphology was restored maximally by combined hPRP/HA treatment, and aggregate and polygonal cell morphology was found (Fig. 2C; Additional file 1: Fig. S1).

Fig. 2

In-vitro 2D hyaluronic acid (HA) and human platelet-rich plasma (hPRP) promote cell activity and anti-inflammatory effects of rat TMJ chondrocyte. Optimal concentration of (A) HA and (B) hPRP dosage in rat TMJ chondrocytes that were analyzed using the MTT assay. C The effect of HA and PRP on the cell numbers and morphological changes (200 ×) of rat TMJ chondrocytes after 2-day treatment with IL1β + TNF-α (I + T)-conditioned medium. I (10 ng/mL) + T (20 ng/mL) were added to the medium to create an in-vitro proinflammatory cytokine-induced arthritic cell model. Real-time PCR analyses of monolayer cultures of rat TMJ chondrocytes after 2-day treatment with I + T-conditioned medium. The results revealed that the combination of HA and hPRP (D) significantly increased cartilage-specific gene expression, including SOX9, collagen type I (Col-I), collagen type II (Col-II), and aggrecan, and E significantly inhibited I + T stimulated inflammatory genes including IL-1β, COX2, MMP-3, and MMP-13. F Immunocytochemistry of Col-II (black arrow indicates presence of Col-II) and its quantification (upper panel) and Alcian blue stain and its quantification (lower panel). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with I + T and control group using paired t test. The results are shown as mean ± SD for three replicate

Combined hPRP/HA therapy modulates gene expression changes in the TMJ-OA in-vitro model

The therapeutic effects of the hPRP/HA treatment were assessed through gene expression analysis of the rat TMJ chondrocytes harvested at 48-h post treatment with I + T. The genes involved in the chondrogenesis process were analyzed. The expression of chondrogenic genes, such as SOX9, Col-II, Col-I, and aggrecan, was significantly decreased, which was restored after treatment with the hPRP/HA (Fig. 2D). Notably, hPRP/HA treated chondrocytes showed significantly reduced expression of proinflammatory genes (IL-1β, COX2, and MMP3) and matrix regulation genes (MMP13); the expression of these genes were enhanced by I + T (Fig. 2E), indicating that the hPRP/HA decreased the levels of activated inflammatory and matrix inhibitory molecules to enhance chondrogenesis. Furthermore, the accumulation of proteoglycan and Col-II in the chondrogenic matrix was determined through Alcian blue and ICC staining, respectively, along with their quantification (Fig. 2F). In the treatment group, the hPRP/HA modulated inflammatory–depleted proteoglycan levels in the cellular OA model, indicating that combined hPRP/HA treatment enhanced the positive signal of Col-II, specifically in the proliferative area (marked with an arrow). However, the group without hPRP/HA treatment exhibited complete degradation of Col-II and proteoglycan compared with the control group.

Combined hPRP/HA therapy promotes chondrocyte regeneration in TMJ-OA in-vitro 3D condyle model

The results from the cellular TMJ-OA 2D model indicated that combined hPRP/HA treatment could strongly modulate inflammation and chondrogenesis to enhance regeneration potential and rescue chondrocyte degradation. To further validate the effects of combined hPRP/HA treatment in an in-vivo mimic microenvironment condition, rat TMJ chondrocyte encapsulated in collagen-derived scaffold of 3D construct with rotary stem cell technique. In our previous studies, we used a cartilage matrix for direct chondrogenesis in a 3D rotary stem cell system [21]. The construct resembles the condyle, with morphological changes observed after 4 weeks of cultivation. The 3D condyle cultured with I + T showed significantly deformed macromorphology, less condylar height, degraded cartilage-like outer surface, and proteoglycan deposition as compared with the control. However, after treatment with the hPRP/HA, the construct exhibited an elastic macromorphology indicating that cell proliferation increased to regenerate chondrocytes to form a condyle, with a smoother cartilage outer surface, improvement in matrix deposition, and condylar height. By contrast, H&E staining revealed higher cell death in the I + T group (Fig. 3A). Similarly, as in the in-vitro model, we conducted Alcian blue and Col-II IHC staining to examine the effect of the hPRP/HA on proteoglycan and Col-II deposition in the chondrogenic matrix, respectively. The results revealed that hPRP/HA-treated 3D condyle cartilage displayed matrix restoration with high proteoglycan deposition (Fig. 3B) and Col-II (Fig. 3C). The hPRP/HA-treated condyle also showed in the anterior, posterior, and central regions of condylar heights (Fig. 3D–G).

Fig. 3

In-vitro 3D hyaluronic acid (HA) and platelet-rich plasma (hPRP) promote cell activity of rat TMJ chondrocytes. For the TMJ-OA model, neocartilage of TMJ chondrocytes/collagen constructs were cultured for 4 weeks in basal control, I + T, and I + T/hPRP/HA -conditioned medium. Constructs were then histologically examined using (A) macromorphology, hematoxylin and eosin (H&E) staining, IHC staining for Col-II, and Alcian blue staining. (B) Percentage areal deposition of s-GAG by quantification of TB-stained area, and (C) percentages of Col-II deposition in TMJ condylar cartilage. (D) Schematic of the anterior, central, and posterior regions of the rat TMJ condylar head. Results (E) posterior, (F) anterior, and (G) central condylar height. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with I + T and control group using paired t test. The results are shown as mean ± SD for three replicate

Collectively, the results for the in-vitro 2D and 3D TMJ-OA disease models demonstrated that combined hPRP/HA treatment promoted TMJ chondrocyte regeneration, inhibited the degeneration of condylar cartilage by reducing proinflammatory factors and matrix regulating factor, and enhanced the expression of the genes for chondrocyte regeneration and matrix restoration of condylar cartilage.

Combined hPRP/HA therapy in TMJ-OA induced animal model

Next, to investigate the therapeutic effect of combined hPRP/HA therapy in an in-vivo model, we first induced TMJ-OA rat through CFA administration at 0 and 14th day and then in the 21st day applied intra-articular injections of the hPRP/HA in the treatment group or PBS in the control group (Fig. 4A). Nociceptive responses were monitored through changes in body weight (Fig. 4B), which showed no significant difference. The head width increased in CFA-induced TMJ-OA was restored by hPRP/HA treatment down reaching to that of the control group at 4 weeks post-treatment (Fig. 4C).

Fig. 4

Experimental design and behavioral evaluation of combined hPRP/HA treatment in CFA-induced TMJ osteoarthritis. A Time course and experimental design. TMJ-OA was induced by two injections of CFA at 0 and 14th day, followed by combined hPRP/HA treatment in the 21st day. CFA and hPRP/HA were administered through the lateral puncture technique. B Body weight changes after treatment with hPRP/HA. C Representative photographs demonstrate that the head width of rats is increased after receiving CFA induction. Both the synovium and disc became thickened, opaque, and head width increased in the CFA-induced TMJ-OA group (marked in black circle) that recovered after treatment in hPRP/HA group. Each group n = 3 rats. *p < 0.05, **p < 0.01, and ***p < 0.001, the results are shown as mean ± SD for three replicates

MRI of TMJ in a TMJ-OA rat after combined hPRP/HA therapy

The therapeutic potential of the hPRP/HA was then evaluated using MRI (Fig. 5A) before and after treatment. At the early point of 1 week, MRI data indicated that no significant difference was observed in the articular disc area, articular cartilage, and temporal bone between hPRP/HA-treated and untreated groups (Fig. 5B, first column). However, after 3 weeks of treatment, the hPRP/HA-treated group exhibited regeneration of both the articular disc area and temporal bone and reduced fibrous thickness of the articular cartilage compared with the CFA-only group. Moreover, after 4 weeks of treatment, compared with the CFA-only group, the condyle in the hPRP/HA-treated group showed complete improvement in the articular disc area, temporal bone, and articular cartilage, which was similar to that of the control group (Fig. 5B, last column). Quantification of the articular disc area also revealed an improvement in a time-dependent manner after treatment with hPRP/HA (Fig. 5C).

Fig. 5

High magnetic field magnetic resonance imaging (MRI) applied to the TMJ in the rat. A Overview of the 7 T micro-imaging system. B MRI scan of control, CFA, and hPRP/HA-treated groups at 1–4 weeks posttreatment. C Measurement of the articular spaces in the coronal view. *p < 0.05, **p < 0.01, and ***p < 0.001 and the results are shown as mean ± SD for three replicates

Combined hPRP/HA therapy reverses TMJ degeneration in TMJ-OA

At 4 weeks post-treatment, the hPRP/HA-treated group exhibited effective restoration of the TMJ condylar structure, with marked improvements in overall cartilage thickness, matrix deposition, cellularity, and condylar height that were comparable to those of the control group (Fig. 6). Compared with the CFA-only group, the hPRP/HA-treated group exhibited reduction in fibrous thickening of the cartilage and enhanced matrix restoration, with higher areal deposition of proteoglycan and Col-II, as revealed by H&E staining (Fig. 6A). Quantification through Alcian blue and Col-II staining showed that the hPRP/HA-treated group exhibited increased matrix and Col-II deposition by 15% and 10%, respectively (Fig. 6B, C). Mankin score was also evaluated for tissue injury. At 4 weeks post-treatment, the Mankin score of the CFA-only group increased deterioration level to 7.5, and the score of the hPRP/HA-treated group further reduced to 3.5, which is close to the Mankin score of the control group (2.0) (Fig. 6D). Moreover, the condyle and cartilage were divided into three main regions: anterior, central, and posterior to be examined (Fig. 6E). Compared with the CFA-only group, the hPRP/HA-treated group displayed increased cartilage thickness in the anterior region (116.4 ± 32 μm), central region (172.9 ± 30 μm), and posterior region (149 ± 42 μm) (Fig. 6F); as well as improvements in the condylar height in the anterior region (779.3 ± 25 µm), central region (1046.8 ± 40 µm), and posterior region (759.7 ± 15 µm) (Fig. 6G). Collectively, the results demonstrated that combined hPRP and HA treatment alleviated TMJ degeneration by suppressing inflammation and pain, reducing adverse fibrosis, and promoting overall matrix restoration of the TMJ condylar cartilage and subchondral bone in later stages of repair. Therefore, the combined hPRP/HA treatment could alleviate tissue injury and enhance tissue repair in TMJ-OA.

Fig. 6

hPRP/HA promote TMJ repair and regeneration in OA. Histopathological evaluation indicated that hPRP/HA restored TMJ matrix synthesis in OA. A Hematoxylin and eosin (H&E), Alcian blue staining, and immunohistochemical staining for Col-II at 4 weeks (black arrow indicate the presence of Col-II). Representative images (n = 3). Scale bars: 500 or 100 μm. B Percentage areal deposition of proteoglycan by quantification of Alcian-stained area, and C percentages of Col-II cells in TMJ condylar cartilage. D Mankin scores of samples at 4 weeks. E Schematic of the anterior, central, and posterior regions of the rat TMJ condylar head. The condylar height was measured at each region, including co-At: anterior height; co-Ct: central height; and co–Pt: posterior height of condyle. The cartilage thickness was also measured at each region, including C-At: anterior thickness; C-Ct: central thickness; and C-Pt: posterior thickness of cartilage. Measurement of F cartilage thickness and G condylar height at the anterior, posterior, and central regions. *p < 0.05, **p < 0.01,, and ***p < 0.001 and the results are shown as mean ± SD for three replicates

hPRP/HA therapy alleviates subchondral bone deterioration in TMJ-OA

The overall reduction in the condylar height with decreased cartilage thickening in the CFA-only group suggested subchondral bone erosion (Fig. 7A). We therefore investigated bone structure changes, including macromorphology, percentage of bone volume (BV), trabecular volume (TV), trabecular number, trabecular thickness, and bone mineral density (BMD) through micro-CT analysis (Fig. 7B). Combined hPRP/HA treatment attenuated bone loss, as indicated by increased BV (Fig. 7C), trabecular thickness (Fig. 7D), and BMD (Fig. 7E). However, relative to the CFA-only group, the hPRP/HA-treated group exhibited a decreased trabecular number (Fig. 7F).

Fig. 7

hPRP/HA treatment restored TMJ subchondral bone volume and architecture in OA. Rat TMJs were harvested for micro-CT analysis at 4 weeks posttreatment of hPRP/HA. A Macromorphological view (white arrow indicates damage area of subchondral bone) and B sagittal view. C BV/TV, D trabecular thickness, E BMD, and E trabecular number. Data represent mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control and CFA group and the results are shown as mean ± SD for three replicates. White arrow indicate damage area of TMJ

Demographic and evaluation parameters for the clinical outcome of hPRP/HA treatment

After the evaluation of combined hPRP/HA therapeutic effect on in-vitro and in-vivo studies, we performed a pilot clinical study in Taipei medical University hospital. In the human clinical trial, we enrolled 20 patients, which were randomly divided into treated group (10 patients) and placebo/non-treated/disease group (10 patients) (Fig. 8). No deaths occurred during the study, and no adverse events led to treatment discontinuation or study termination. TMJ-OA patient was administered with the hPRP/HA injections (Fig. 9A), the patients’ condition were evaluated using MRI, visual Analogue scale (VAS), and examination of Maximum Mouth Opening (MMO), and Activity of Daily life (ADL).

Fig. 8

Consort flowchart for clinical trial of hPRP + HA in TMJ-OA disease

Fig. 9

Therapeutic potential of the combine hPRP/HA treatment in the clinical study. A Schematic of HA and PRP administration into the superior space of TMJ-OA patients. B Disease group without treatment at 0 and 6 months of oblique sagittal MRI images represent disk displacement and osteoarthritis become worse. Quantification of the disc displacement distance from the condyle (C) and condylar thickness were measured at 0 and 6 months (cases 1 and 2, n = 1) (D). In the treatment group (E), patients of disc displacement with reduction (DDR), disc displacement without reduction (DDWR) are designated as case 1, and case 2, respectively, that were associated with disk degeneration, and osteoarthritis of the condyle at 6 months post-treatment. Oblique sagittal MRI images indicated disc recapture and condylar remodeling after using the hPRP/HA treatment compared with the disease or untreated group. In the treatment group disc displacement distance from the condyle (F) and condylar thickness (G) was measured at 0 and 6 months. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with I + T and control group using paired t test

MRI screening of the TMJ after hPRP/HA therapy in patients with TMJ-OA

MRI images were evaluated before and after treatment for disc displacement, disc degeneration, condylar bone changes as signs of bone degenerative process, or disk perforation. In case of disc displacement with reduction (DDR) in the closed mouth position, the posterior region of the disk is anterior to the condyle, whereas in the open mouth position, it returns to the normal position. However, in case of disc displacement without reduction (DDWR) in the closed mouth position, the posterior region of the disk is anterior to the condyle, and the disc size is normal, whereas in the open mouth position, the posterior region of the disk cannot return to its normal position between the condyle and articular eminence [24]. In the disease group/non-treated, MRI images at 0 and 6 months revealed that disc displacement (Fig. 9B) and its distance from the condyle remained unchanged (Fig. 9C); however, the condyle was degraded, and its thickness was reduced (Fig. 9D). By contrast, in the treatment group, after 6-month MRI data revealed slight restoration of disk displacement in DDR cases 1 and DDWR case 2 (Fig. 9E). Moreover, in both cases, disc displacement toward the condyle occurred (Fig. 9F), decreasing the disk displacement distance and increasing the condyle thickness to restore the shape of the condyle (Fig. 9G).

Clinical assessment after hPRP/HA therapy in TMJ-OA patients.

After the treatment of hPRP–HA, patients were followed up to 6 month (Additional file 1: Table S1), and improvement in pain was assessed using VAS. VAS pain scores significantly decreased to 2.7 at 1 month and after 6 months the VAS pain score is 0 compared with 4.5 score in patients with TMJ-OA (Fig. 10A) (Table 2; Additional file 1: Tables S2, S3.1, and S3.2).

Fig. 10

Clinical findings of hPRP/HA treatment. A Comparison of pain (VAS score) during the follow-up period with the preoperative level of pain. A greater decrease in pain was observed in the hPRP/HA-treated group. B Comparison of MMO during the follow-up period with preoperative MMO. A progressive increase in MMO was observed in the hPRP/HA-treated group. C Comparison of ADL during the follow-up period with the preoperative level of ADL was also increased in hPRP/HA-treated group. Clinically, the MMO and ADL scores were significantly increased in the hPRP/HA-treated group compared to the control (non-treated/disease) group, and the pain (VAS) score was significantly decreased. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared within hPRP + HA group using paired t test

Table 2 VAS result for the control and hPRP + HA study group

Patients with TMJ-OA are also not able to open their mouth properly. Therefore, we also evaluated the MMO based on the parameters in Table 3 (Additional file 1: Tables S4, S5.1, and S5.2). MMO was recorded in cm, and results revealed a significant increase in MMO after treatment with hPRP/HA (Fig. 10B). Patients with TMJ-OA have pain, reduced MMO, and decreased quality of life, thus considerably reducing their ADL, such as eating, talking, and laughing. ADL were measured in terms of scores of 0–25 (Table 4; Additional file 1: Tables S6, S7.1, and S7.2). We observed that after the treatment with hPRP/HA, the ADL score increased as compared to patients with TMJ-OA (Fig. 10C).

Table 3 MMO result for the control and hPRP + HA study groupTable 4 ADL result for the control and hPRP + HA study group