Nasal septum-derived chondroprogenitor cells control mandibular condylar resorption consequent to orthognathic surgery: a clinical trial

Abstract Condylar resorption is an aggressive and disability form of temporomandibular joint (TMJ) degenerative disease, usually non-respondent to conservative or minimally invasive therapies and often leading to surgical intervention and prostheses implantation. This condition is also one of the most dreaded postoperative complications of orthognathic surgery, with severe cartilage erosion and loss of subchondral bone volume and mineral density, associated with a painful or not inflammatory processes. Because regenerative medicine has emerged as an alternative for orthopedic cases with advanced degenerative joint disease, we conducted a phase I/IIa clinical trial (U1111-1194-6997) to evaluate the safety and efficacy of autologous nasal septal chondroprogenitor cells. Ten participants underwent biopsy of the nasal septum cartilage during their orthognathic surgery. The harvested cells were cultured in vitro and analyzed for viability, presence of phenotype markers for mesenchymal stem and/or chondroprogenitor cells, and the potential to differentiate into chondrocytes, adipocytes, and osteoblasts. After the intra-articular injection of the cell therapy, clinical follow-up was performed using the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) and computed tomography (CT) images. No serious adverse events related to the cell therapy injection were observed during the 12-month follow-up period. It was found that autologous chondroprogenitors reduced arthralgia, promoted stabilization of mandibular function and condylar volume, and regeneration of condylar tissues. This study demonstrates that chondroprogenitor cells from the nasal septum may be a promise strategy for the treatment of temporomandibular degenerative joint disease that do not respond to other conservative therapies.


Introduction
Degenerative joint diseases (DJD) begin as cartilage erosions or loss of subchondral bone mineral density, associated with inflammatory processes, which may be its trigger or immunologic consequence. 1 This process can be characterized as osteoarthritis, a progressive disease of the entire joint organ, 2 with significant morbidity worldwide.Osteoarthritis may attack different synovial joints, including the temporomandibular joint (TMJ).The major clinical diagnostic criterion for TMJ intra-articular inflammation is the painful limitation of mouth opening. 3However, cases of subclinical osteoarthritis with a degenerative joint disease but without arthralgia can also occur, and their monitoring should be done by the followup of images.
TMJ computed tomography (CT) showed that cortical erosions and subchondral cysts are the main characteristics that differentiate patients from asymptomatic controls. 4A longitudinal study with a 5-year follow-up demonstrated that subchondral cysts tend to form in areas under osteoarthritis mechanical overload, mostly leading to bone resorption and deformation of the mandibular condyle. 5Studies of the natural course of severe TMJ osteoarthritis also showed that most discontinuous cortex erosions are converted to continuous ones for up to 2 years.However, there is also an increased probability of condylar volume reduction by resorption, 6 even when they are conservatively treated 7 The concept of condylar resorption (CR) has now gained attention.Whether this is a separate disease or a subtype of TMJ osteoarthritis has yet to be determined.The evidence of different etiologies of condylar resorption is relatively low, and its clinical features are comparable to those of severe degenerative joint diseases. 6This resorption is one of the significant post-surgical complications of orthognathic surgery, and the resorption degree of condylar height is related to post-operative skeletal relapse. 8Risk factors for condylar resorption following orthognathic surgery include disc displacements and degenerative lesions before treatment 9 especially when mandibular advancement associated with counterclockwise rotation is done.The incidence of condylar resorption following mandibular osteotomies varies between studies, reaching up to 31%. 10 Different protocols have been proposed to address the effects of condylar resorption after orthognathic surgery.They range from pharmacological therapies 11 to surgical procedures involving the repositioning of the articular disc 12 or even the total TMJ prostheses replacement. 13Regenerative medicine has proven to be an alternative for orthopedic cases involving advanced degenerative joint disease, and the knee has served as a translational research model for developing innovative TMJ therapies. 14ue to its avascular nature, 15 the fibrocartilage covering the mandibular condyle has a limited capacity for self-repair.Mesenchymal progenitor cells reside in the outermost condyle layer and constitute the only cell reservoir able to participate in cartilage self-regeneration.During such processes, fibrocartilage chondroprogenitor stem cells' proliferation, differentiation, and maturation are regulated by complex signals involving the Wnt/β-catenin pathway. 16However, the maintenance of non-physiological joint overload can lead to abnormal activity of this pathway and induce unexpected chondrocyte hypertrophy, leading to cartilage degeneration through increased expression of multiple catabolic factors. 17he use of the nasal septum as a source of cartilage mesenchymal stem cells has been previously described. 18In a firstin-human trial, these cells were harvested, matrix engineered, and applied to repair knee cartilage defects, efficiently integrating the grafted cells into the adjacent native cartilage and the underlying subchondral bone. 19Our group has proposed a novel treatment for condylar resorption after orthognathic surgery using autologous nasal septum stem cells transplantation and reported the first human case. 20In the present study, we describe the results of a phase I/IIA clinical trial evaluating the safety and efficacy of autologous chondroprogenitor cells derived from the nasal septum for the treatment of patients with TMJ condylar resorption associated with orthognathic surgery.

Patients
The present study has been approved by the Petrópolis Medical School/ UNIFASE Committee of Ethics in Research (CEP-FMP/UNIFASE) and by the National Commission of Ethics in Medical Research (CONEP), CAAE 12484813.0.0000.5245.The study was retrospectively registered in the Brazilian National Clinical Trials Registry and the US Clinical Trials Registry under the Universal Trial Number (UTN) U1111-1194-6997.Inclusion criteria: Patients aged 18 or over, diagnosed with dentofacial deformities related to severe degenerative disease of the TMJ, irrespective of the presence or absence of concomitant arthralgia, and with a surgical indication for orthognathic surgery to correct the dentofacial deformity.Exclusion criteria: pregnant or breastfeeding patients, patients with local infection or other comorbidities that contraindicate the surgical procedure, rheumatic diseases, and chronic pain in a joint other than the temporomandibular Patients were enrolled after signing an informed consent form.After indicating the regenerative medicine approach and enrollment in the present study, 10 research participants underwent a nasal cartilage biopsy to isolate and process chondrocytes derived from the nasal septum.This procedure was performed during the orthognathic surgery, under general anesthesia, and 20 mL of blood was collected from the patient to prepare autologous serum.All steps of this study followed a systematic timeline (Supplementary Figure S1).
The present open-label phase I/IIA clinical study aimed to evaluate the safety and efficacy of autologous chondroprogenitor cells derived from the nasal septum for treating patients with TMJ condylar resorption.This study is the first globally to administer these cells into the temporomandibular joint, its design involved a limited number of patients, and the gradual inclusion of participants was recommended by the National Research Ethics Commission (CONEP).The inclusion followed a careful scheme, starting with 2 patients, and the absence of complications within 3 months allowed the progressive inclusion of additional patients.After observing the absence of serious adverse events, we expanded the study to a total of 10 patients.
Ten participants were included, 9 received intra-articular injection of autologous chondroprogenitor cells, in both TMJs, while 1 participant (ID 8) could not be treated due to an in vitro cell contamination.Participants were recruited between January of 2016 and October of 2021.The average age of participants was 30.6 years; 7 females and 3 males.Participants were followed up for 12 months, being submitted to clinical evaluation using the DC/TMD for TMJ signs and symptoms.

Nasal septum cartilage processing and quality control
Nasal cartilage biopsy processing, cell culture conditions, cell suspension for therapy, and cell transport conditions were prepared as described previously. 20All quality controls were performed on the final product before infusion.Sterility was evaluated by tests to detect bacteria and fungi (Bact/Alert 3D, Biomerieux), endotoxins (Endosafe PTS, Charles River), and mycoplasma (KIT MycoAlert PLUS for Mycoplasma Detection, Lonza).Cell viability controls were done by flow cytometry using the vital dye 7-aminoactinomycin D (7-AAD; BD #559925), to determine the percentage of viable cells, and Annexin V protein (BD #51-65,875X) to determine the percentage of cells in apoptosis.Cytogenetic analysis was done by the GTG-banding.
To evaluate the potential for cell differentiation into adipocytes and osteoblasts, cells were plated in triplicates (40 000 cells/well) in 24-well plates on glass coverslips to monitor their potential adipogenic and osteogenic differentiation, according to. 21Their potential of chondrogenic differentiation was assessed by micromass culture.Approximately 1.5 × 10 6 cells in 1 mL of culture medium were centrifuged at 300g for 10 minutes in a conical tube to form a cell pellet.The collagen type II deposition areas were quantified using Image Pro-Plus 4.5 software (Media Cybernetics, Rockville, MD, USA), and the areas were converted to percentages.

RNA extraction, cDNA synthesis, and quantitative PCR
After the chondrogenic differentiation, the cell aggregates were resuspended in TRI Reagent (Sigma) followed by addition of chloroform.After centrifugation, the aqueous phase was isolated, mixed with 70% ethanol and added to the purification columns from RNA isolation with PureLink RNA mini kit (Thermo Fisher), and the manufacturer's instructions were followed.For each sample, 132 ng of RNA was used for the cDNA synthesis using the ImProm-II Reverse Transcription System (Promega), again according to the manufacturer's instructions.cDNA amplification was done with GoTaq qPCR Master Mix (Promega) and the specific primers listed in Supplementary Table S1, in a final volume of 10 µL.Quantitative PCR (qPCR) was done in the QuantStudio 5 Real-Time PCR system (Thermo Fisher) and the results were analyzed with the QuantStudio Design and Analysis Software version 2.6.0 (Thermo Fisher).The Cq results for each gene were normalized based on RNA polymerase II (POLR2) expression.
The results were plotted as a relative expression using the 2 −ΔΔCt (fold change), comparing induced and non-induced cells and as the expression ratio to RNA polymerase II (POLR2) expression (fold to POLR2).

Clinical injection of cells
Cells from passage 3 to 5 were suspended in phosphatebuffered saline (PBS), supplemented with 25% (v/v) of injectable solution containing 10 mg/mL sodium hyaluronate (Osteonil mini, TRB Pharma) and 5% autologous serum.The final volume injected in each TMJ was 1 mL containing 10 7 cells after arthrocentesis, according to procedures described by De Souza Tesch et al. 20

Clinical follow-up
The same examiner carried out the clinical evaluation of the patients of this study at the time of diagnosis (initial clinical evaluation), and during the clinical follow-up period at 7 and 15 days, 1, 3, 6, and 12 months after application of the treatment, using "Diagnostic Criteria for Temporomandibular Disorders" (DC/TMD) for TMJ signs and symptoms.

Images acquisition and condylar remodeling analysis
The images were acquired in a Cone Beam CT "I-Cat Classic" (Imaging Science International ) using the extended height protocol: field of view 16 × 22 cm, scan time 40 s, and voxel size 0.4 mm.The acquisition protocol was the same at T1 and T2 (obtained before and up 15 months post therapy, respectively) to avoid differences in image resolution.The acquired images were saved in DICOM file format.The ITK-SNAP 3.8.0software (www.itksnap.org) 22was applied to the segmentation step, and 3D slicer 5.0.2 software (www.3dslicer.com) 23 to register the scans and their respective 3 dimensional volumetric models, following the protocol used by De Souza Tesch et al 20 .
The visual analysis of 3-dimensional morphological and volumetric changes was done, using 3D Slicer 5.0.2 software with the extensions modules ModelToModelDistance and ShapePopulationViewer, which allow the measurement of the distance between two 3-dimensional models and to visualize and compare the surfaces at the same time.
These modules illustrate the difference between distances of 3-dimensional models (T1 e T2) through color variation, considering that green areas show some degree of resorption between the initial and final models, a yellow color identifies moderate changes, and a red color identifies marked differences between models indicating bone apposition.
After co-registration, both baseline and follow-up geometries were sliced within the same plane, removing any discrepancy in the original slicing.Then, for each geometry, the volume was computed based on the face normal values, using Blender 3.5 software, a 3D modelling and rendering package, (http://www.blender.org).The method was proposed by Zhang and Chen 24 and allows the computation of the volume of a triangulated mesh using solely the triangle vertices.

Results
The nasoseptal chondroprogenitor cells were positive for CD105, CD73, and CD90 surface markers, described for mesenchymal progenitor cells of different origins 25 (Table 1).The CD44 hyaluronan receptor that organizes the pericellular matrix was also highly expressed in the majority of nasoseptal chondrogenic progenitor cells from all the research participants.Unexpectedly, the nasoseptal chondrogenic progenitors were positive in vitro for CD146 (passages 3 to 5) (Table 1), a perivascular cell marker first described in the bone marrow. 26We observed CD146 + in over 90% of the expanded nasal septum cells in the later passages (3 to 5), which were injected into the research participants.As an exception, only participant ID 9, 61 years old, had only half of the analyzed cell population positive for CD146 (50.8%) (Table 1).At the same time, the cluster of differentiation markers that usually characterize stem cells of the hematopoietic origin (CD14, CD19, CD34, and CD45) was nearly absent (Table 1).The samples exhibited expression of type II collagen both prior to and subsequent to the initiation of chondrogenic differentiation, demonstrating their commitment to the chondrogenic lineage, as shown in Figure 1A.The quantitative evaluation of collagen type II immunopositive cells revealed no statistically significant distinction between the groups subjected to chondrogenic induction and the control (Figure 1F).Although nasoseptal chondroprogenitor cells were committed to the chondrogenic lineage, they were able to differentiate into adipocytes or osteoblasts when induced into these lineages (Figure 1B and Figure 1C).Cytoplasmic lipid droplets were accumulated in vitro in cultures of some human nasoseptal chondrogenic progenitor cells (Figure 1B).Similarly, nasoseptal chondrogenic progenitor cells could deposit considerable extracellular calcium under an osteogenic-inducing medium (Figure 1C).For adipogenic and osteogenic differentiation, the absorbance was quantified (Figure 1D and Figure 1E), and a statistically significant difference (P < .01) between cells induced to adipogenic or osteogenic differentiation and the not induced ones showed the ability of chondroprogenitor cells to respond to the specific induction.
The gene expression profile of nasoseptal chondroprogenitors (qPCR) showed that they increased their type I and type II collagen expression when differentiating in vitro into the chondrogenic lineage (Figure 2).The analysis of SOX9 expression showed that there is no difference between the induced and non-induced conditions after 21 days of cell culture (P < .05; Figure 2).This may indicate that the profile of their transcription factors may have already been defined in the analyzed cell culture passage and differentiation time.The same profile was observed for RUNX2, with no statistically significant difference to the control (P < .05),indicating that the analyzed cells could not reach a hypertrophic phenotype at this stage (Figure 2).Also, it is interesting to note that there is some variation in the gene expression between different cell donors (Supplementary Figure S2).
The main initial clinical complaints were severe or moderate TMJ arthralgia, TMJ crepitus, limited pain-free mouth opening, occlusal instability (5 out of 9 participants), crepitus (6 of 9 participants), and mandibular function limitation (5 out of 9 participants) 12 months after cell therapy injections, participants presented a decrease of arthralgia levels, improvement in mandibular function, increased maximum pain-free mouth opening, and absence of the TMJ crepitus (Supplementary Table S2).

Medical complications during the clinical trial
In our clinical study, adverse events were systematically classified into 3 categories: non-serious, moderate, and severe, aligning with international clinical research guidelines and CNS Resolution no.466, of October 12, 2012, Brazil.
We did not observe any severe adverse event related to autologous chondroprogenitor cells injection.A total of 23 adverse events were observed: 14 mild ones (mild TMJ pain, vertigo, masticatory muscle pain, night sweats, hyperhidrosis, purulent secretion on teeth), and 9 moderate ones (TMJ pain, headache, and gastritis) (Supplementary Table S3).The most frequent adverse reactions were TMJ arthralgia, masticatory muscular pain, and headache.Arthralgia and myalgia must probably be related to the injection procedure itself.It is noticeable that in TMJ pain has been improved over the follow-up period in all the research participants.
All events were recorded on a specific form.Our medical and dental team monitored each one until their resolution.Subsequently, the treatments administered were analyzed for their effectiveness in resolving health problems.All adverse events were promptly reported to the Research Ethics Committee (CEP) and submitted to the National Research Ethics Commission (CONEP).The events underwent a thorough evaluation by CONEP, and the clinical study continued as no serious adverse events were reported."

Autologous chondroprogenitor cells therapy reduced arthralgia
We used 2 pain assessment methods from the DC/TMD: (1) characteristic pain intensity and (2) chronic pain severity (Von Korff Scale). 27utologous cell therapy reduced the pain intensity reported by all participants, with clinical improvement in the characteristic pain intensity over time (Table 2).All the participants reported pain improvement, and most of them considered that pain was absent, just 1 month after the experimental treatment.At the 6-month clinical follow-up, 78% of participants reported no pain.At 12 months after cell transplantation, 7 of the participants (78%) considered the pain absent, 2 (22%) considered the intensity as low (< 50), and no one as high (≥ 50).
The chronic pain severity analysis using the Von Korff Scale, also showed a reduction in the degree of pain-related disability after treatment.Before the chondroprogenitor cells injection, most participants considered the pain as high intensity (grade II) or with severe related disability (grade IV).One month after cell transplantation, most of participants considered joint pain absent (n = 6).At 12 months after cell injection, no research participant reported pain considered as grade II (high intensity), III or IV (with related disability) (Table 2).

Autologous chondroprogenitor cells therapy promoted the stabilization of mandibular function
We observed that the therapy using autologous progenitor cells promoted the stabilization of the mandibular function.Among the 9 participants included, 4 had previous mandibular dysfunction (one intense, 2 moderate, and one mild degree; Supplementary Table S4).One month after the autologous cell therapy, the participant ID 5, with the worst and most severe mandibular dysfunction, completely improved and never presented it again.The 2 patients (ID 1 and 2) with moderate dysfunction also improved.The increase in the maximum range of pain-free mouth opening and maximum assisted mouth opening (Table 3), in addition to the stability of the clinical status in most of the participants, are evidence that the treatment prevented the progression of the TMJ degenerative disease, despite the overload produced by the orthognathic surgery procedure.
There was a clinical improvement in almost all participants (88%, n = 8) concerning maximum pain-free mouth opening when comparing before and 12 months after treatment.Half of the participants were below the cutoff point (≥ 35 mm) at the time of inclusion in the clinical study and reached an opening range above this point after treatment.In the first month after cell injection, some participants showed a decrease in pain-free mouth opening amplitude.However, this impairment was possibly related to the orthognathic surgery and not to the cell injection procedure.A comparative analysis between the clinical segments showed that, from the first month onwards, all participants showed an improvement in the maximum pain-free mouth opening compared to the previous clinical segment (Table 3).Similar results were observed in the maximum assisted mouth opening analysis.4 research participants below the expected average (≥ 40 mm) had a progressive improvement until the end of the follow-up period (Table 3).

Autologous chondroprogenitor cells promoted joint tissue regeneration
Analyzing the initial and final CT scans (acquired 8 to 15 months after the therapy injection) (Table 4) and volumetric evaluation of the mandibular condyles before and after ACT (Supplementary Table S5), we could observe the following phenomena: recorticalization without volume change; completed regeneration, evidenced by recorticalization with volume increase; regeneration in progress, in which there is volume gain but without final recorticalization; stabilization, where cortical and volume are maintained; adaptive remodeling, with loss of volume leading to the recovery of  Clinical assessment of chronic pain severity, using the Von Korff scale, which classifies pain as absent (0), grade I, grade II, grade III, and grade IV.
Table 3. Clinical analysis of pain-free maximum mouth opening and maximum assisted mouth opening.
joint space without discontinuous cortical in this specific region and therapeutic failure with discontinuous cortical structure and loss of volume.Although these phenomena can simultaneously occur in different areas of the same condyle, there is generally a predominance of one of them in the joint as a whole.Figure 3 shows images categorized based on the predominant phenomena observed in the selected condyles, with 2 condyles allocated to each group: CT scans with the respective 3D reconstructions and superimposition of the pre-treatment and post-treatment models, showing the areas of apposition of bone tissue (positive values).The intensity of the orange or red color is directly related to the amount of new bone tissue formed (Figure 3).The intensity of yellow or green is directly related to tissue balance or resorption.Areas with a value next to zero indicate a balance between apposition and resorption (Figure 3).Tissue regeneration promoted by chondroprogenitor cells was shown by cortical continuity and new areas of bone tissue on CT images.In addition, in the 3-D reconstructions, the areas of bone regeneration are indicated by color intensity of positive values (Figure 3).

Discussion
Most currently available conservative treatment strategies for CR focus only on symptom management 28,29 with limited success and not addressing or halting disease progression 30 Currently, TMJ CR cases do not respond to conservative or minimally invasive therapies, and frequently evolves to surgical intervention and prostheses implantation. 28owever, ideally, TMJ CR treatment should aim not only at relieving pain and restoring function but also preventing the progression of cartilage and subchondral bone loss, targeting orthopedic stability.Biological cartilage repair techniques are emerging, with methods such as autologous transplantation of chondrocytes or chondroprogenitor cells showing encouraging results. 31Our group recently reported the first case of intra-articular injection of cultured autologous cells from nasal septum to control TMJ CR, with CT images evidencing articular bone regeneration. 20esearchers still deal with the challenge of chondrocyte expansion in vitro. 32Isolation and expansion of cartilage cells probably involve at least chondrocytes and their progenitors.The different obtention protocols for each cell type and their  membrane markers after expansion are still controversial. 32he cells injected in this clinical trial were isolated following the protocol described by Amaral et al, 18 which obtained chondroprogenitor cells from the nasal septum fibrocartilage perichondrium cambium layer with surface markers characteristic of mesenchymal progenitor cells.Cells obtained from cartilage tissue after culture passages 33 or in an inflammatory environment, 34 were demonstrated to suffer dedifferentiation, starting to express clusters of differentiation (CD) characteristic of mesenchymal stem cells (MSC). 33They also start to synthesizing collagen I 34 and displaying multi-potent stem/progenitor cell characteristics, 33 such as anti-inflammatory and immune-modulatory activities.So, they could suppress the injury-induced production of inflammatory mediators within joint tissues, which are responsible for at least part of clinical symptoms. 35MSCs preferentially attracted to and harbored in diseased tissue rather than intact tissue.When intra-articular injected, they bind not only into cartilage but also bone defects, proliferate and participate in their regeneration. 36he expression of CD146 increases after subsequent culture passages, as verified as part of the dedifferentiation process in other studies. 33,37As described elsewhere 18 by part of our research group, in the first passages of the nasal septum chondroprogenitors subculture (one to 3), cells were positive for surface markers described for mesenchymal stem cells, except CD146.The absence of this perivascular cell marker is consistent with their avascular niche in fibrocartilage.On the other hand, CD146 + cells can also recreate a hematopoiesissupportive human bone-like tissue 26,38 supported a developmental hierarchy of skeletal progenitors in humans where a purified population of PDPN+ CD146-CD73+ CD164+ cells serially generate colony forming units from single cells in vitro and multi-lineage ossicles containing bone, cartilage, and stroma upon sub-renal transplantation in mice.
Therefore, it is still unclear whether fibrocartilage stem cells (FCSC) are quiescent stem cells that are left from earlier development, remain reserved in tissue, and then become reactivated upon in vitro culture or, instead, whether most mature chondrocytes have such potential to become progenitors.
Most of the nasal septum cultured cells (≥ 90%) injected into 8 of the 9 research participants expressed CD146 membrane marker in the latter passages (3 to 5).As an exception, 49.2% of the analyzed cell population, at passage 4, of research participant ID 09, a 61-year-old woman, was negative for CD146.With increasing age, a decrease in the dedifferentiation capacity of MSC has already been verified, 39 and also in the expression of the CD146 marker.This was probably reflected in the behavior of the isolated and injected cells of this specific research participant, which promoted the higher volume of tissue formation of the entire sample (45.33%).The possible combination of different cell types (chondroprogenitor cells and dedifferentiated chondrocytes) within the injury site can lead to a synergistic action between them, simultaneously forming cartilage and bone through different growth processes.
Furthermore, the cells isolated presented a statistically significant increase in type II and type I collagen expression in the group submitted to chondrogenic differentiation compared to the control group.Interestingly, the cells of participant ID 09, expressed high levels of SOX9 and type 2 collagen in control group compared to induced group (Supplementary Figure S2).
This different expression pattern may be related to distinct cell populations that behave differently to the same stimulus.
All these characteristics and behaviors of nasal septum progenitor cells may control the progression of DJD and potentially promote the formation of new tissue.DJD are better classified through CT images, regardless of painful symptoms, being also fundamental in the follow-up of the disease's natural history or response to treatment. 7Qualitative and quantitative volumetric analyzes were previously performed to assess the stability of severe TMJ OA in its natural course.The presence of a continuous cortex determined the absence of notable volumetric losses at a mean 2 years follow-up. 6lthough most areas of cortical erosion (74.4%) converted into continuous cortex during this period, the mean volume loss was close to 15%.Mandibular condyle cortical line continuity and density were also related to condylar volume changes after orthognathic surgery.Considering a loss of condylar volume of less or equal than 10% as a sign of stability, the continuous cortex group showed a higher stability rate (92.5%) compared to the discontinuous cortex group (51.3%). 6n this clinical trial, analyzing 8-15 months CT images follow-up, it was possible to observe the formation of appositional tissue at the upper surface of the mandibular condyles, either with initially discontinuous cortical, hypodense but continuous cortical, or normal continuous cortical.The exceptions were condyles presenting continuous cortex but deformity in these regions.Analyzing the initial and final volume of the 6 condyles with a gain of volume, we observed a medium volume increase of 21.33% (Supplementary Table S5).Both bone apposition and resorption areas are considered when evaluating the volume, and even when condyle volume was considered stable, bone apposition was noticed.Apposition was observed in the superior and posterior surfaces, commonly subjected to bone resorption.It was possible to identify areas of apposition very close to or precisely in the same areas of previous cortical discontinuity (Figure 3).Our hypothesis for this new tissue formation is that the cortical discontinuity or its low density allowed the contact of the injected cells with the vascular niche of the bone marrow, which may have led the cartilage progenitor cells to differentiate into osteoblasts.Direct contact between FCSC and endothelial cells significantly enhanced their osteogenic differentiation. 40On the other hand, when the injected progenitor cells do not establish direct contact with the endothelial cells, they remain at the cartilage lesion's surface, forming cartilaginous tissue that can later be replaced by bone tissue through a process resembling endochondral ossification. 41FCSC is also shown to be able to form transient cartilage and initially inhibit angiogenesis to generate an avascular cartilage.Later, proangiogenic growth factors may be released to promote the proliferation of endothelial cells and form bone tissue. 40nother possibility is direct transdifferentiation, where hypertrophic chondrocytes could directly differentiate into osteoblasts and form bone. 42 The hypothesis that CR after orthognathic surgery is part of a progressive process of pre-surgical resorption was previously evaluated by 3D condylar remodeling.The condylar volume during the presurgical phase was considered relatively stable (−3.3 ± 37.2 mm 3 ).However, condylar volume suffered a significant reduction during the post-surgical phase, on an average of −12.2%. 43In this clinical trial, 12 condyles decreased their volume after orthognathic surgery despite cell therapy injection.However almost all of them (8 of 12) presented a volume loss lower or equal than 10%, which was previously considered as stability of volume for such cases. 6The average percentage of loss observed in this clinical trial, considering 4 condyles, was 27.82% (Supplementary Table S5).
In terms of safety, the outcomes of the reported cases were consistent with previous studies using culture-expanded autologous cells and administered via intra-articular route the managing DJD in a various other pathologic conditions. 44he procedure was generally well tolerated with only a mild and brief increase in discomfort noted immediately after injecting the therapy.No cell-related serious adverse events or abnormalities in laboratory parameters or clinical signs were observed, potentially being a safe and valuable therapy if evidence accumulate.
The results suggest that autologous cartilage progenitors can become a suitable treatment for the control of TMJ arthralgia and related disability secondary to CR, with possible regenerative properties of joint tissues.This therapeutic approach should be considered in patients refractory to traditional conservative treatments before escalating to more invasive surgical interventions.

Conclusion
The reported therapies proposed for the treatment of temporomandibular degenerative diseases show that a single intraarticular injection of autologous chondrocytes, expanded in vitro and expressing some of the markers of cartilage progenitors, was safe and well tolerated during the 12-month follow-up period.Using clinical and image analyses, we report improved arthralgia and mandibular function, and favorable structural improvements in TMJ tissues affected by condylar resorption.Further investigations should accumulate sufficient evidence to propose new strategies to treat temporomandibular degenerative diseases refractory to other therapies.

Figure 1 .
Figure 1.(A) In vitro chondrogenic differentiation.Cells were induced to chondrogenic differentiation (induced) or were not induced (control).Type II collagen immunohistological staining.(B) In vitro adipogenic differentiation.Cells were induced to adipogenic differentiation (induced) or were not induced (control).Oil Red O dye identifies the presence of lipid vacuoles inside the cells.(C) In vitro osteogenic differentiation.Cells were induced to osteogenic differentiation (induced) or were not induced (control).Brightfield and corresponding Alizarin Red stained identify calcium deposits produced by differentiated cells.(D) Percentage of type II collagen immunopositive cells.No significant difference between the control group and the cells induced to differentiation was observed.(E) Oil Red O quantification and (F) Alizarin Red S quantification.After differentiation, samples were submitted to the Oil Red O dye absorbance quantification assay to quantify the adipogenic differentiation and the Alizarin Red S dye absorbance quantification assay to quantify osteogenic differentiation.A statistical difference was observed for adipogenic and osteogenic differentiation (P < .01).Scale bar= 200 μm (A) and 50 μm (B and C).

Figure 2 .
Figure 2. Relative expression analysis of genes related to chondrogenesis and osteogenesis after chondrogenic differentiation.The dotted line represents the expression at control (non-induced) condition.Statistical analysis was performed using the Mann-Whitney test.*P < .05.

Figure 3 .
Figure3.Image analysis by computed tomography of the TMJ joint and 3-dimensional remodeling images of condyles of participants before and after autologous chondroprogenitor cells joint injection.Right and left CT images from the patients before (upper line) and 8-15 months after (bottom line) the treatment: external lateral view (0 °), frontal view (90 °), and internal lateral view (180 °).Prior to the initiation of the treatment, specific regions were demarcated using arrowheads, with the color blue designating instances of discontinuous cortical structure, and rose indicating continuous cortical structure.Subsequent to the treatment, the designated areas were redefined with arrows, wherein blue represented bone apposition and recorticalization, red signified bone apposition, white denoted recorticalization, rose represented continuous cortical structure, and yellow was assigned to discontinuous cortical structure.Additionally, the presence of a blue star was used to highlight areas undergoing adaptive remodeling.The color map indicates condylar resorption (negative value) and apposition (positive value) on the superimposed condylar surfaces (before and after treatment models).The orange arrows establish a correlation between the pre-treatment and post-treatment regions in the computed tomography (CT) scans and their respective counterparts observed in the 3-dimensional superimposed images.

Table 2 .
Clinical evaluation of joint pain during clinical follow-up.

Table 4 .
Analyses of TMJ by CT exam before and after (8 to 15 months) ACT injection.