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Combination of polyetherketoneketone scaffold and human mesenchymal stem cells from temporomandibular joint synovial fluid enhances bone regeneration
Therapies using human mesenchymal stem cells (MSCs) combined with three-dimensional (3D) printed scaffolds are a promising strategy for bone grafting. But the harvest of MSCs still remains invasive for patients. Human synovial fluid MSCs (hSF-MSCs), which can be obtained by a minimally invasive needle-aspiration procedure, have been used for cartilage repair. However, little is known of hSF-MSCs in bone regeneration. Polyetherketoneketone (PEKK) is an attractive bone scaffold due to its mechanical properties comparable to bone. In this study, 3D-printed PEKK scaffolds were fabricated using laser sintering technique. hSF-MSCs were characterized and cultured on PEKK to evaluate their cell attachment, proliferation, and osteogenic potential. Rabbit calvarial critical-sized bone defects were created to test the bone regenerative effect of PEKK with hSF-MSCs. In vitro results showed that hSF-MSCs attached, proliferated, and were osteogenic on PEKK. In vivo results indicated that PEKK seeded with hSF-MSCs regenerated twice the amount of newly formed bone when compared to PEKK seeded with osteogenically-induced hSF-MSCs or PEKK scaffolds alone. These results suggested that there was no need to induce hSF-MSCs into osteoblasts prior to their transplantations in vivo. In conclusion, the combined use of PEKK and hSF-MSCs was effective in regenerating critical-sized bone defects.
Cells used in this study were isolated from human synovial fluid of TMJ from TMD patients. In our pilot studies, hSF-MSCs of healthy volunteers were obtained and isolated, but these were not used in the current study. Those MSCs from healthy donors shared similar flow cytometric characteristics to MSCs from TMD patients. However, the main difference was that hSF-MSCs could only be isolated from ~16.7% of healthy donors TMJ synovial fluid45. Also, the number of colony forming units (CFUs) of hSF-MSCs from healthy donors at plating (passage 0) was much lower, and this resulted in an insufficient cell number for our subsequent experiments. Another reason for using hSF-MSCs from TMD patients was the availability of the synovial fluid following the arthrocentesis treatment; while synovial fluid collection would not have been indicated for healthy patients (i.e. without TMD). Furthermore, our long-term goal is to use autologous hSF-MSCs to reconstruct the mandibular condyle of patients suffering from severe TMJ osteoarthritis, and thus this was another reason for selecting hSF-MSCs from TMD patients for this study. hSF-MSCs displayed typical MSCs characteristics based on their cell surface markers (CD73, CD90, CD105, CD45, etc), multipotency, and self-renewal capacity (Fig. 1). In our previous study about hSF-MSCs34,46,47,48, Flow cytometric experiments were done from the third to sixth passages of hSF-MSCs. At passage 6th, more than 96% of hSF-MSCs were positive for MSC markers and less than 2% of hSF-MSCs were positive for (CD45, CD34, CD11b, CD19, and HLA-DR). These flow cytometric results were not statistically different when compared to those cells at passages 3rd to 5th. CD44 is a receptor for hyaluronic acid, which is a major component of articular surfaces49. Previous reports focused on the chondrogenesis function of hSF-MSCs and their potential applications in cartilage repair24,26,35. hSF-MSCs were also reported to have comparable biological characteristics with BMSCs and possessed a greater osteogenic potential than other MSCs obtained by non-invasive procedures, such as the dental pulp or exfoliated deciduous teeth stem cells23,26. However, their contributions in regenerating bone defects have barely been investigated in the literature. By testing the osteogenic ability of hSF-MSCs on 3D-printed scaffolds both in vitro and in vivo, our study proposed an additional source of non-invasively harvested human MSCs for bone regeneration as well as an optimal source of autologous cells to repair bone defects of TMD patients.
The first finding of this study was that the surface of 3D-printed PEKK scaffolds was biocompatible for cell attachment and growth. PEKK belongs to the polyaryletherketones family (PAEKs) which are materials with mechanical properties that can coexist with human bone37. PEKK is considered a promising material to replace metals and ceramics currently used in orthopedics36,38,50 and in dentistry42,43,51,52,53. However, there are only a few studies36,39 reporting the effect of PEKK on stem cells. This study tested a 3D-printed PEKK scaffold with 750–1000 µm interconnecting channels. The laser sintering process resulted in a rough and porous surface, when observed at the SEM level (Fig. 2a,d). This surface topology might have favored cell attachment and their osteogenic differentiation. Although hSF-MSCs proliferated slower on PEKK than on tissue culture plastic (TCP; 2D culture), cells on PEKK showed multiple filopodia and lamellipodia (Fig. 2b,e,f). Both structures are composed of actin fibers and transmembrane adhesion complexes. Filopodia act as sensors to explore the extracellular environment whereas lamellipodia can induce cell migration by supporting traction forces from the cell actomyosin network54,55. These images (Fig. 2b,e,f) suggested that the surface of our 3D-printed PEKK had adhesive interactions with the seeded cells and might modulate cellular signalings toward osteogenesis.
The second finding of this study was that the PEKK scaffold by itself could not induce osteogenesis in hSF-MSCs but required the osteogenic cultured media. As expected, hSF-MSCs cultured on either PEKK or TCP, and in non-osteogenic media did not show an osteogenic differentiation. However, when hSF-MSCs were cultured in osteogenic media, this cultured condition enhanced the osteogenic differentiation of hSF-MSCs as the result shown in groups PEKK + OS and TCP + OS (Fig. 3). ALP activity levels were higher in hSF-MSCs grown on PEKK than on TCP, as early as day 4 and day 7 (Fig. 3a). Also, an upregulation of osteogenesis-related genes was detected at day 21 for cells grown on PEKK (Fig. 3b). ALP is a transient early marker of osteogenic differentiation for MSCs56, indicating their ability to differentiate into osteoblasts57. An initial rise in ALP activity, with a peak at day 14, was observed in PEKK + OS and TCP + OS groups, which was in line with several previous studies57,58,59. This observation was confirmed with the downregulation of the ALP gene at day 21. Unlike ALP, other osteogenic genes such as OPN, OCN, COLІA1, and RUNX2, all exhibited a significant upregulation when cultured in osteogenic media for 21 days. These markers were commonly used in many other studies to demonstrate the osteogenic potential of MSCs, and whether the MSCs were at their early or late stage of osteogenic differentiation. To obtain an overall ability of hSF-MSCs in maintaining their osteogenic phenotypes when cultured on PEKK scaffolds, the mRNA levels of these markers were measured at day 21, which was a widely accepted timepoint in the literatures. RUNX2 encodes a key transcription factor with an essential role in the commitment of MSCs towards osteoblasts60. However, its expression pattern during osteogenesis varies. Huang X et al.61 reported that the pattern of RUNX2 expression fluctuated during osteogenic differentiation of MSCs. The expression of RUNX2 was found to increase initially and then to decrease in human dental pulp MSCs, while it remained consistently high in human bone marrow MSCs during their osteogenic/odontogenic differentiation61. It was also reported that the mRNA level of RUNX2 correlate positively with the level of OCN during osteogenesis62. Therefore, we measured both the osteogenic late markers and their upstream regulator (RUNX2) to allow us to detect their correlations. The mRNA level of RUNX2 was significantly higher in the PEKK + OS group. Not surprisingly, the expression of OCN on PEKK was 5.8 folds higher than the TCP + OS group, suggesting an enhanced mineralization of the extracellular matrix (ECM) and more active osteoblasts. ECM provides a template for mineralization, which mainly consists of collagens (especially collagen type І)63. A higher expression of COLIA1 indicated a more active formation of ECM on PEKK relative to TCP. The highest upregulated gene that we measured during hSF-MSCs culture was OPN. This indicated a proliferation of pre-osteoblasts in vitro as OPN is secreted prior to matrix mineralization64. The increased gene expression levels of OPN, OCN, COLІA1, and RUNX2 implied that hSF-MSCs cultured on PEKK 3D-scaffolds were induced into an accelerated osteogenic differentiation in vitro. Such benefits of PEKK (i.e. increased mRNA levels toward osteogenesis) along with its biocompatibility and biomechanical properties confirmed its use as an adequate scaffold for bone tissue engineering.
The third finding of this study was that (naïve/non-differentiated) hSF-MSCs were more effective in regenerating bone when compared to osteogenically-induced hSF-MSCs. Our in vivo results demonstrated that the volume of bone formed (~20%) by hSF-MSCs was twice higher than the bone volume (~10%) of osteogenically-induced hSF-MSCs. This was a surprise to us because some researchers had previously reported that it was better to osteogenically-induced MSCs prior to their in vivo transplantation to increase bone regeneration17,65. We hypothesized that the undifferentiated/naïve hSF-MSCs possessed a greater cell plasticity and responded to the rich cocktail of healing stimulus in the critical-sized defects16,17,66. Nevertheless, the decision to transplant naïve/non-differentiated versus osteogenically-induced MSCs to increase bone formation remained controversial16,17,65,67. This discrepancy might be due to studies using MSCs from different tissues, different scaffold materials, and different animal models. Results from this study indicated that there was no need to osteogenically-induced human MSCs from TMJ synovial fluid when cultured on PEKK prior to their in-vivo implantation into the rabbit cranial critical-sized defect model.
Last but not the least, our in-vivo results supported the use of PEKK as a scaffold for hSF-MSCs. Our micro-CT and histological results (Figs 5 and 6) revealed a distinct increase in bone regeneration when either hSF-MSCs (PEKK + SF) or osteogenically-induced hSF-MSCs (PEKK + OS) were seeded on PEKK scaffolds. According to our histological analysis, non-differentiated hSF-MSCs (from treatment group PEKK + SF) formed larger and more matured osteoid, while osteogenically-induced hSF-MSCs (from treatment group PEKK + OS) produced fewer and smaller mineralized tissue, which was mainly located to the periphery/border of the critical-sized defect (Fig. 6). However, newly-formed peripheral bone still contributed to the integration of PEKK into the critical-sized defect because results from the Push-Out Strength test of both PEKK + SF and PEKK + OS groups were higher than those from the PEKK group (that had no seeded hSF-MSCs) (Fig. 7). Histologically, we did not detect an increased number of inflammatory cells when PEKK was transplanted with hSF-MSCs. This result suggested no (observable) immunological rejection of the xenogeneic (human) hSF-MSCs in the rabbit model. Our observations were in agreement with earlier studies supporting the immunomodulatory properties of human MSCs68. Still, the rabbit calvarial site might be more immune-privileged69 and we could not exclude this possibility. On the contrary, Adamzyk et al.36 found no added effect of transplanted ovine BMSCs on bone regeneration in sheep calvarial defects. Adamzyk and colleagues reported, in their flow cytometric data, a lower percentage of MSCs from ovine bone marrow. Also, the sheep model mounted an immunological response to transplanted allogeneic cells36. These two factors might explain a decreased potential of bone regeneration observed by these authors.
Although the bone defect was not completely healed within the 12-week duration of this study, the regenerated bone in the group that was implanted with PEKK seeded with hSF-MSCs demonstrated a functional recovery. Additional studies with a longer follow-up period are required to investigate whether complete closure of the bone defect would be established with our proposed grafting material combined with hSF-MSCs. Also, to further confirm the benefits of PEKK scaffolds, additional studies comparing PEKK to other typical bone scaffolds will be needed.
In conclusion, hSF-MSCs from the TMJ could be easily harvested with minimal invasiveness to the patients. hSF-MSCs possessed high multipotency and could be an alternate cell source to BMSCs for bone regeneration, as well as an optimal autologous cell source for TMJ repair. The combination of hSF-MSCs and PEKK (a biocompatible bone-mimetic polymer) resulted in an increased osteogenic ability, both in vitro and in vivo. The combined implantation of PEKK and hSF-MSCs is a promising therapy to regenerate large bone defects.
source:https://www.nature.com/articles/s41598-018-36778-2
