- ICH GCP
- US Clinical Trials Registry
- Clinical Trial NCT04998058
Autogenous Mesenchymal Stem Cell Culture-Derived Signalling Molecules as Enhancers of Bone Formation in Bone Grafting
Study Overview
Status
Conditions
Intervention / Treatment
Detailed Description
Aims and Hypothesis
Based in previously reported studies and following a translational approach, a hypothesis that the paracrine effect linked to cultured autogenous growth factors and cytokines at physiologic concentrations acting locally in grafted sites might promote a faster and/or more efficient cell response and consequently induce a more significant/faster bone formation was considered. The presence of these molecules added to synthetic bone substitutes might act positively in terms of local cell recruitment (chemotaxis), proliferation, differentiation and bone protein synthesis. Also, recurrent biological hazards involved in conventional tissue-engineered cellular graft compounds, usually linked to the ex vivo manipulation of cells in terms of tumorigenicity, immunogenicity and the previously reported cell dedifferentiation phenomenon would be mitigated by the exclusion of the cell element of this process, as they are considered by some reports to present unpredictable mitotic behavior once artificially cultured. The present study aims to further extend this translational investigation following preclinical studies by the principal investigator, proposing a prospective randomized controlled clinical trial on the possible benefits associated with the application of a tissue-engineered bone graft compound containing concentrated autogenous cell-cultured medium (CM) and an hydroxylapatite/beta-tricalcium phosphate-based synthetic bone substitute (HP/β-TCP ceramic). Specific aims include analyses on the density of the newly formed calcified tissue by computed tomography (CBCTs), expression of specific immunohistochemical bone formation markers (RT-PCR) and histomorphometric bone quantity evaluation. In a split-mouth study model, the resultant bone formation after HP/β-TCP grafting with and without CM will be analyzed, compared and quantified at different time points.
- Materials and Methods
2.1- Study design
A prospective split-mouth randomized controlled clinical trial was chosen as study design for the present investigation. After protocol approval by the Research Ethics Committee and registration at the clinical trials.gov platform, following CONSORT guidelines, 20 consecutive patients who seek maxillary oral rehabilitation at the Department of Prosthetic Dentistry of the School of Health and Life Sciences - PUCRS, Brazil, will be invited to participate in the present investigation.
2.2 - Lipoplasty and human adipose stem cells (hASCs) harvesting/culture.
For hASCs harvesting, an abdominal lipoplasty procedure will be proposed and performed following a signed informed consent. Under intravenous sedation and supplementary oxygen release with an oral catheter, along with local anesthesia, a standard surgical lipoplasty technique will be performed. As previously described, from this liposuction material, 25 to 30 ml of fat will be transferred into a 50 ml Falcon tube. The fat will be then washed by centrifugation (430Å~g 10 min), being the upper fat layer transferred into a new Falcon tube, where an equal volume of collagenase solution will be added to the mixture and incubated at 37°C in a water bath. After centrifugation, the upper fat layer will be discarded and the supernatant removed. The remaining cell pellet containing the hASCs will be resuspended with 15 ml of culture medium. After adding 15 ml of warm culture medium, the cell suspension will be transferred into a T175 culture flask. The hASCs will be incubated in a humidified environment at 37°C and 5% CO2. Then, the cell pellet will be resuspended in warm culture medium and seeded into new cell culture flasks.
2.3 - Concentrated culture medium (CM) preparation and analysis
Stem cell culture and CM preparation will be performed at a GMP certified facility for regenerative medicine products. At first, the surface antigen profiles of isolated hASCs at third passage will be characterized by flow cytometry. The presence of CD73, CD90, CD105 and CD44 markers and the absence of CD34, CD45, CD11b, CD19 and HLA-DR will be assessed to confirm the desired cell phenotype as recommended by protocols of the International Society of Cellular Therapy.[ Then, a sample of these cells will be also characterized by staining (Alizarin Red S) in accordance to the manufacturer's instructions. Cell expansion and CM will be generated following standard stem cell culture protocols. Before clinical use, CM will be examined not only for contamination with bacteria, fungi, or mycoplasmas but also for infection with viruses including hepatitis B and C, human immunodeficiency and human T-cell leukemia viruses. Then, CM will be properly cool-stored and sent to clinical application (up to 6 hours), to avoid long-term denaturation/inactivation of present signalling molecules, as previously suggested.
2.4 - Maxillary sinus floor elevation procedure
Under IV sedation and supplementary oxygen release with an oral catheter, along with local anesthesia, the maxillary bone will be grafted internally by drilling an access in the lateral maxillary sinus wall. Sinus membrane dissection with careful release and elevation will be also performed. Preoperative randomizing by computer-generated random numbers will be used to determine test and control sinuses for each patient. The bony floor of the maxillary sinus on the test site will be augmented with 4 to 5g of a synthetic bone substitute (BoneCeramic™ 1-2 mm) mixed with 10 to 15 ml of CM. The control site will receive similar amount of bone substitute embedded in 10 to 15 ml of saline solution. Patients will be released from hospital facility 2h after surgery. They will be instructed in postoperative hygiene and eating behavior. All patients will receive postoperative antibiotic and anti-inflammatory therapies.
2.5 - Implant placement and bone biopsies harvesting
After 6 months of healing, implant placement will follow routine surgical protocols as recommended by the implant manufacturer. Under local anesthesia, a mid-crestal linear incision and full-thickness flap will be performed with releasing incisions whenever necessary. Then, with a 16:1 contra-angle attached to a surgical unit and a trephine bur (Ø 3mm), the grafted area correspondent to the 2nd premolar, 1st and 2nd molar regions will be biopsied at both right and left sides of the maxilla (n=06 sites per patient), guided by a previously made acrylic resin surgical stent. Bone biopsies will be then divided in two segments, where one will be stored in 4% formaldehyde solution for histologic analysis and the other in Eppendorf vials with RNALater for RT-PCR analysis. Then, the sequence of implant surgical burs as recommended by the manufacturer will be followed for the placement of 6 to 8 implants per patient. Registration of primary stability at insertion will be done by both manual torque wrench and classified into three groups. A healing abutment will be then placed, and wound closure will be conducted with non-resorbable suture material A 6-month one-stage healing protocol will be adopted for all implants.
2.6 - CBCT image analysis
High-resolution CBCT images will be obtained at three different time-points as part of the treatment protocol, meaning at pre-operative bone graft [baseline], 90 [T1] and 180 [T2] days (implant placement pre-operative), aiming both pre-op surgical planning and post-op evaluation of the bone formation. The morphometric bone parameters will be calculated in 3D analysis according to the recommendations of the American Society for Bone and Mineral Research (ASBMR) as previously proposed, and statistical analysis will be used to identify the best parameter combinations aiming to differentiate trabecular bone into three bone categories: (i) sparse-related to a loose bone structure, (ii) intermediate-related to a well-structured trabecular bone, and (iii) dense bone types-related to a massive bone area with little space between the trabeculae.
2.7 - Histologic and histomorphometric analyses
Following removal of graft biopsies at implant placement, bone blocks containing the control and test bone sites will be shaped and decalcified with 5% HNO3. The blocks will dehydrate in a graded alcohol series, clarify with xylene and be embedded in resin. Then, the resin will be polymerised in a UV light chamber for 10h. Using a diamond micro saw, a total of three 3-μm-thick slices from each block will be ground transversely to each specimen long axis at 50, 100, and 150μm from their external portion. After this, routine staining with hematoxylin-eosin (HE), Azur II and Pararosaniline will be done aiming to differentiate between HP/β-TCP particles and newly formed bone. Microscopic analysis will be performed at 24X magnification using an optical stereo microscope connected to a digital camera. Image analysis will be performed using the ImageJ® software.
2.8 - Real-time polymerase chain reaction (RT-PCR)
Following the preparation of the collected graft biopsies for RNA extraction, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis will be applied to quantify alkaline phosphatase, vascular endothelial growth factor, Osteonectin, Osteopontin and type 1 collagen.
Study Type
Enrollment (Estimated)
Phase
- Phase 2
- Phase 1
Contacts and Locations
Study Contact
- Name: Eduardo R Teixeira, DDS, Ph.D.
- Phone Number: +555133203562
- Email: eduardo.teixeira@pucrs.br
Study Contact Backup
- Name: Rosemay A Shinkai, DDS, Ph.D.
- Phone Number: +555133203562
- Email: rshinkai@pucrs.br
Study Locations
-
-
RS
-
Porto Alegre, RS, Brazil, 90619-900
- Pontificia Universidade Católica do Rio Grande do Sul
-
-
Participation Criteria
Eligibility Criteria
Ages Eligible for Study
Accepts Healthy Volunteers
Description
Inclusion Criteria:
- patients >35 years-old.
- presenting highly atrophic fully edentulous maxilla (residual bone height <5 mm).
- In need of bilateral sinus floor augmentation aiming full mouth implant-supported rehabilitation.
- having teeth extraction at least 8 weeks prior to bone augmentation.
Exclusion Criteria:
- smokers, illicit drug users and alcohol daily consumers.
- patients with metabolic and/or systemic diseases leading to impaired healing (e.g. decompensated diabetes, leukocyte or coagulation disorders, immunosuppression).
- history of radiotherapy in the head or neck region.
- bisphosphonate-based therapy recipients.
- intolerant to general/local anesthesia.
Study Plan
How is the study designed?
Design Details
- Primary Purpose: Other
- Allocation: Randomized
- Interventional Model: Parallel Assignment
- Masking: Double
Arms and Interventions
Participant Group / Arm |
Intervention / Treatment |
---|---|
Placebo Comparator: Control (conventional graft procedure), bone substitute and saline solution.
one randomly assigned maxillary sinus grafted internally defined as control (bone substitute + saline solution) per subject.
|
Bone augmentation at the floor of the maxillary sinus with bone substitutes.
Other Names:
|
Experimental: Test (modified graft), bone substitute and concentrated culture medium (CM)
one randomly assigned maxillary sinus grafted internally defined as test (bone substitute + concentrated culture medium) per subject.
|
Bone augmentation at the floor of the maxillary sinus with bone substitutes.
Other Names:
|
What is the study measuring?
Primary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
Assessment of changes in bone density and quantity
Time Frame: 0 (baseline), 90 and 180 days
|
Comparative analysis (test vs. control) of the changes in bone density and quantity by cone beam computed tomography (CBCT).
|
0 (baseline), 90 and 180 days
|
Assessment of bone quantity
Time Frame: 180 days
|
Histomorphometry analysis expressed in percentage of newly formed bone
|
180 days
|
Secondary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
Presence of bone formation markers
Time Frame: 180 days
|
RT-PCR assessing specific bone formation markers
|
180 days
|
Other Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
---|---|---|
Assessment of bone substitute resorption rate
Time Frame: 180 days
|
Resorption rate of bone substitute particles (test vs.control) compared by histologic analysis and histomorphometric analyses.
|
180 days
|
Collaborators and Investigators
Collaborators
Investigators
- Principal Investigator: Eduardo R Teixeira, DDS, Ph.D., Pontificia Universidade Católica do Rio Grande do Sul
- Study Chair: Denise C Machado, MSc., Ph.D., Pontificia Universidade Católica do Rio Grande do Sul
- Study Chair: Rogerio B Oliveira, DDS, Ph.D., Pontificia Universidade Católica do Rio Grande do Sul
- Study Chair: Rosemary A Shinkai, DDS, Ph.D., Pontificia Universidade Católica do Rio Grande do Sul
- Study Chair: Patricia Sesterheim, MSc, Ph.D., Institute of Cardiology of Rio Grande do Sul
- Study Chair: Reinhilde Jacobs, DDS, Ph.D., KU Leuven
Publications and helpful links
General Publications
- Boeckel DG, Sesterheim P, Peres TR, Augustin AH, Wartchow KM, Machado DC, Fritscher GG, Teixeira ER. Adipogenic Mesenchymal Stem Cells and Hyaluronic Acid as a Cellular Compound for Bone Tissue Engineering. J Craniofac Surg. 2019 May/Jun;30(3):777-783. doi: 10.1097/SCS.0000000000005392.
- Cabreira CL, Fulginiti RL, Sesterheim P, Shinkai RSA, Teixeira ER. Effect of hyaluronic acid on paracrine signaling of osteoblasts from mesenchymal stromal cells: potential impact on bone regeneration. Clin Oral Investig. 2021 Jul;25(7):4571-4578. doi: 10.1007/s00784-020-03771-x. Epub 2021 Jan 7.
- Teixeira ER, Boeckel D, Fulginiti R L, Shinkai R S A, Machado D. Mesenchymal stem cells and hyaluronic acid for bone grafting. Clinical Oral Implants Research, 2018, (29), 55-55.
Study record dates
Study Major Dates
Study Start (Estimated)
Primary Completion (Estimated)
Study Completion (Estimated)
Study Registration Dates
First Submitted
First Submitted That Met QC Criteria
First Posted (Actual)
Study Record Updates
Last Update Posted (Actual)
Last Update Submitted That Met QC Criteria
Last Verified
More Information
Terms related to this study
Additional Relevant MeSH Terms
Other Study ID Numbers
- Bone Graft-Exosomes Project
Plan for Individual participant data (IPD)
Plan to Share Individual Participant Data (IPD)?
IPD Plan Description
Drug and device information, study documents
Studies a U.S. FDA-regulated drug product
Studies a U.S. FDA-regulated device product
This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.
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