Prospective Clinical Study with New Materials for Tissue Regeneration: A Study in Humans

Abstract Objective  This study was performed to evaluate the clinical, radiographic, and histomorphometric outcomes of novel bone grafting materials and dental membranes and to compare the results with current data from the literature. Materials and Methods  New synthetic bone substitutes, consisting of biphasic calcium phosphate in the ratio of 60% hydroxyapatite and 40% β-tricalcium phosphate, were applied in bony defects and covered by either a novel synthetic poly(lactic-co-glycolic) acid (PLGA) or porcine collagen membrane. A sample of 51 biomaterials was placed in a total of 20 patients during different surgical protocols. Implants were simultaneously inserted, and in the case of sinus floor elevations 6 months later. Pre- and postoperative cone-beam computed tomographies were taken. Bone biopsies were harvested from augmented sides and processed for histomorphometric evaluation. Statistical Analysis  Averages and ranges were calculated for the percentage of newly formed bone, residual biomaterial, and connective tissue. Data were submitted to analyze the radiological mean differences in length, width, and density. Paired t -tests were deployed for the analysis of differences within each group between the baseline (preoperative) and the final (postoperative) measurements. Results  The mean bone gain in length and width were 0.96 ± 3.33 mm (+27.59%) and 1.22 ± 1.87 mm (+30.48%), respectively. The bone density was increased by a factor of 4, reaching an average of 387.47 ± 328.86 HU. Histomorphometric evaluations revealed new bone formation of 41.44 ± 5.37%, residual biomaterial of 24.91 ± 7.31%, and connective tissue of 33.64 ± 4.81%. The mean healing period was 8.32 ± 3.00 months. Conclusions  Data from this study confirmed the suitability of the tested materials in dental surgery. The biomaterials may be recommended for various clinical procedures. A satisfactory level of increase of new bone was reported in augmented sides. No significant differences were observed between the tested membranes. PLGA membranes might be superior to collagen membranes for their easier handling.


Introduction
The biggest challenge facing treatments in implantology and maxillofacial surgery is achieving an adequate bone volume as well as a successful osteointegration for long-lasting results.Dental bone defects may be caused by trauma, genetic anomalies, cancer, age-related diseases, and in most cases as a natural physiologic process after tooth loss or extractions. 1Thus, partial or complete edentulism leads in the corresponding region to bone loss and soft tissue changes which in turn mean unfavorable conditions for future prosthetic rehabilitation.Accordingly, the reconstruction of these alveolar ridge defects is indispensable by means of bone augmentation techniques, commonly assisted by bone substitute materials.Conventional bone grafts of biological origin are autografts (autogenous bone), allografts (homogenous bone), and xenografts (heterogeneous bone).4][5][6] The most common and commercially offered alloplasts are calcium phosphate (CaP)-based graft substitutes or bioactive glass ceramics. 2,6,7CaP materials are represented by hydroxyapatite (HA), αand β-tricalcium phosphate (α-TCP and β-TCP), and biphasic calcium phosphate (BCP), a mixture of HA and β-TCP.][8][9][10][11] Synthetic bone grafts have many beneficial qualities, including no risks of disease transmission, unlimited supply, biocompatibility, lower morbidity, and predictability. 1,7,9The biological performance of alloplastic biomaterials depends on many factors such as chemical composition, morphology (for example, granule size, porosity, crystallinity), or mechanical stability. 2,3,9These properties can be modified by thermal (sintering methods) or chemical treatments, 6,9,12 thus, bone regeneration can be directly influenced. 9,13Generally, synthetic materials are described as having an osteoconductive behavior 3,4,7 with an osteoinductive potential. 14Bone graft substitutes should resemble, in terms of macro-and microporosity structures, the natural human bone. 12Porosity allows carrying out biological and biochemical processes like bone cell ingrowth, protein adsorption, and vascularization, and accordingly, interactions of the biomaterial surfaces with the surrounding tissue. 4,15,16In guided bone regeneration (GBR), bone graft substitutes are combined with barrier membranes to maintain the stability of the augmented bone volume.Ideally, membranes should guide the slower migrating bone cells to the defect side, while preventing epithelial tissue ingrowth into the graft side. 1 In general, membranes can be classified as resorbable collagen membranes of porcine or bovine origin, synthetic resorbable poly(lactic-coglycolic) acid (PLGA), and polylactide or nonresorbable expanded polytetrafluoroethylene membranes.Nowadays, numerous patients favor nonautogenous, synthetic bone graft substitutes due to ethical, cultural, or religious reasons. 17he present study was focussed on the synthetic bone substitute granules (TIXXU GRAFT; Bredent Medical GmbH & Co. KG, Senden, Germany) and the synthetic bone putty (TIXXU GRAFT, Bredent Medical GmbH & Co. KG, Senden, Germany), coverd by either a synthetic PLGA membrane (TIXXU CONTROL, Bredent Medical GmbH & Co. KG, Senden, Germany) or a porcine collagen membrane (EZ Cure, Biomatlante, Vigneux-de-Bretagne, France).The hypothesis was that the tested materials have similar results to those described in the current literature suitable for the clinical procedures proposed.

Ethical Research in Humans
All patients involved had provided their informed consent prior to inclusion in the study.The experiments were performed under the guideline established by the Declaration of Helsinki as revised in 2008 for medical research involving humans.Possible side effects or complications would be immediately treated in accordance with current medical university knowledge.The study and all associated documents were approved by the Ethical Committee of the Catholic University of Murcia (UCAM).

Study Design
Patients were recruited at the Department of Master's Degree in Implant Dentistry at the Dental Clinic of UCAM, and were treated from March 2018 to December 2020 within this project.The present study was an analysis of the records of 20 systemically healthy patients (7 females and 13 males), partially or completely edentulous, and finally restored with implants for prosthetic rehabilitation.The mean age of the patients was 54.09 AE 11.26 years (ranging from 38 to 75) at the time of surgery.The follow-up period was set as a minimum of a half year.In total, 35 clinical acts have been implemented, and 51 samples of the study material were used.
Conclusions Data from this study confirmed the suitability of the tested materials in dental surgery.The biomaterials may be recommended for various clinical procedures.A satisfactory level of increase of new bone was reported in augmented sides.No significant differences were observed between the tested membranes.PLGA membranes might be superior to collagen membranes for their easier handling.
This Study Included the Following Tested Materials  4,16 Data from in vitro studies 18 showed that the main elements of this material were calcium and phosphate in a ratio of approximately 1.5 to 2.2.The material had a total and intraparticle porosity of 74 and 32%, respectively.The pore size and distribution were around 65 to 72%, and the particle density was approximately 3.2%.The biomaterial had a micro-(<10 µm) and macroporous (100-600 µm) structure with documented grain sizes between 0.5-1 and 0.8-1.5 mm in diameter.The resorption rate was approximately 6 months.The following material was the fully synthetic membrane (TIXXU CONROL, Bredent Medical GmbH & Co. KG, Germany), made of PLGA.The tested membrane had a double-layered structure, fabricated in two processes of freezing and lyophilization with a PLGA solution. 19The outer side of the membrane was covered by a smooth fascia of a dense glossy layer to prevent the ingrowth of gingival fibroblast cells and connective tissue (CT) invasion.The inner layer of the membrane had a porous matt fascia structure with nonwoven microfibers for the promotion of osteogenic cells and a controlled bone regeneration.The bilayer transmembrane structure allowed angiogenesis.
According to the manufacturer's specifications, the resorption rate was around 6 months.The second membrane (EZ Cure, Biomatlante, France) had a cross-linked structure, originally from porcine.It was a commonly recommended collagen membrane for GBR with a documented resorption rate of around 3 months. 1The used size of both membranes was 20 mm Â 30 mm.

Preoperative Examinations
Potential patients were informed about study conditions.All patients received careful periodontal examinations, including the assessment of supra-and subgingival plaque, gingivitis, probing depth, followed by oral hygiene instructions and, if indicated, periodontal therapy.The clinical cases were documented preoperatively, during the surgery, and postoperatively with full-HD pictures (camera: Canon EOS 200D, Japan; macro ring flash lite system: Meike MK-14EXT; macro lens 105 mm: Sigma F2.8 EX DG OS HSM, Japan).

Radiological Analysis
Standardized three-dimensional radiographs were obtained by means of a paralleling cone-beam computed tomography (CBCT) device (Orthophos SL 3D, Dentsply Sirona, United States/Germany), using a digital imaging software system (SIDEXIS 4, Dentsply Sirona).CBCT scans were taken before and at a minimum of 6 months after the surgery.For comparing the bone volume changes, parameters such as the length, width, and density of the recipient graft side were analyzed, first without, and finally with the applied material by using a dental planning software (Blue Sky Plan 4, United States).The alveolar bone of maxilla and mandible was measured in their entire length (basal-occlusal distance) and width (bucco-palatal/lingual distance) at three points.Density was measured in four random points.Always the same pre-and postoperative cross-sectional views were used for analysis in CBCT scans.

Surgical Protocol
All surgeries were performed under local anesthesia, according to the manufacturer's protocol, in the maxilla (n ¼ 16) and mandible (n ¼ 5).Depending on the locus of the bony defect, five different clinical treatments were performed: sinus floor elevation (n ¼ 6), bone regeneration in the third molar region (n ¼ 1), socket prevention (n ¼ 15), ridge augmentation (n ¼ 11), and crest splitting (n ¼ 2).The materials were applied in the bone defects to augment and increase the bone volume; cases in combination with membranes, n ¼ 14, and without, n ¼ 6.The needed quantity of the materials was previously calculated in a patient's protocol, including the adjusted diameter, length, and amounts of the implants (Bioner Implant Systems, Barcelona, Spain), and a further prosthetic rehabilitation plan.In all clinical situations, implants were immediately inserted at the bone crest level, expected during the sinus lift augmentation.After the elevation of the sinus membrane, the implants were placed in the healed bone at least in 6 months' time.In total, 57 implants were placed during the study period in either the augmented sides or areas which were in close contact with biomaterials in the same jaw.After the surgeries, each patient received an anti-inflammatory treatment: 400 mg of ibuprofen every 8 hours for 3 days, and 0.12% chlorhexidine gel every 12 hours for 2 days.Patients were asked to follow the general guidelines after surgical procedures.Temporary prosthetic restoration was done.Sutures were removed after 8 to 10 days postoperative.

Histologic Processing
Bone biopsies were taken with trephine needles after 6 months from the placement of biomaterials, processed for ground sectioning.In total, 11 biopsies were harvested.The protocol started with the fixation process.Samples were dehydrated in increasing grades of ethanol from 70 up to

Histomorphometry
The digital quantitative analysis was performed by using calibrated digital images, ranging from 4Â to 40Â magnification (Leica microscope Q500Mc, Leica DFC320s, 3,088 Â 2,550 pixels, Leica Microsystems, Barcelona, Spain).The most central sagittal section of each implant was taken for histomorphometric analysis using MIP 4.

Statistical Analysis
Results were transferred into Excel (Microsoft Corp., Redmond, Washington, United States).Data analysis was performed with SPSS 20.0 software (New York, United States).
The descriptive method was used to analyze the radiological mean differences in length, width, and density.Quantitative variables in the form of mean, median, standard deviation, maximum, and minimum were calculated for radiologic and histomorphometric evaluations.The paired t-test was deployed for the analysis of the differences within each group between baseline (preoperative) and final (postoperative) measurements.A p-value of <0.05 was established to be statistically significant.

Results
The values followed a normal pattern of dispersion with a 95 percent confidence interval.►Table 1 summarizes the statistically significant differences (p < 0.05) between the measurements of the parameters pre-and postoperative.
The univariate analysis showed a bone gain in length, width, and density of 27.59%, 30.48%, and 315.66%, respectively.

Operation Day
In 76% of the cases, biomaterials were applied in the maxilla, and in 24% in the mandible, whereby their distribution was incidental.The planned prosthetic rehabilitations after substitute grafting and implant insertion were either a fixed hybrid acrylic prosthesis or crowns and/or bridges.All crowns and bridges were of metal-ceramic reconstruction.

Postoperative Situation
Seven of the 57 implants were lost in nonaugmented areas.In all other cases, uneventful healing was observed.Neither regional bony infections nor rejection of the biomaterials could be identified in clinical and radiographic examinations.Regarding the membranes from a clinical point of view, PLGA membranes were rigid under dry conditions, thus, easier to cut into the appropriate shape to cover the bony defects.After moistening, the mechanical flexibility increased, and a simple placement was possible.In comparison, the collagen membrane was softer, and stuck slightly at the instruments.The permeable cross-linked collagen structure was beneficial in allowing cell attachment from all sides, Attention should be paid to place the porous side of the PLGA membrane correctly on the bone surface without confusions of the sides.While suturing with soft tissue, the mechanical strength of both membranes seemed to be enough.The tested membranes did not disturb tissue healing or bone healing as demonstrated in ►Fig. 1.

Radiographic Evaluation
No signs of osteolysis were observed.resorption of the material, resulting in a release of calcium and phosphate, and an increase of these elements in the newly formed bone that is close to the substitute. 6,9Osteoclasts have the same behavior at the implantation side as at the bone surface.High extracellular calcium levels stimulate osteogenesis in tissue by attracting osteoblasts.A successful osteointegration includes the replacement by autologous tissue after biomaterial degradation. 6In vivo studies by Yamada et al 21 confirmed that BCP (60% HA/40% β-TCP) might be seen as the satisfactory mixture for the clinical application.HA operated for a longer time as a mechanical strong scaffold regarding its slow 1 to 2 years resorption rate, 1,4,21 whereas β-TCP was approximately 10 to 20 times faster resorbed due to its higher calcium concentration during the first 3 months. 1,22The properties of the combination of both materials were comparable to the biological degradation rate of natural human bone. 238][29] One disadvantage of autogenous bone is the higher rate of bone formation initially, but in turn also a faster resorption rate. 301 Accordingly, based on the present study, one might conclude that augmentation with alloplasts, in the form of granules and/or putty, is recommendable with a confirmed satisfactory outcome up to a height of 3 to 4 mm.For augmentations with synthetic particulate bone graft higher than 4 mm, further clinical studies are necessary.Regarding the membranes, some studies underlined better outcomes for GBR using barrier membranes, 32 and others showed no significant differences. 33In the present study, the results of radiographic and histomorphometric evaluations according to the PLGA and collagen membrane did not differ substantially, and both membranes had good outcomes as physical and biological barriers.In general, no systemic toxicity has been observed yet for PLGA membranes, 34 and collagen membranes are cytocompatible because of their natural origin. 35oornaert et al 36 figured out that without using additonal bone grafting materials, PLGA membranes have a higher bone regeneration rate of 30% NB, compared with crosslinked porcine collagen membranes (24.6%).PLGA was hydrolyzed after 6.5 months, whereby collagen membranes were already completely resorbed in around 8 weeks.Collagen membranes have faster degradation rates depending on different factors, including the tissue of origin or mechanical properties. 36Alveolar bone healing was marked at around 4 months 36 ; therefore barrier membranes should sustain its biological function during this period.Consequently, PLGA membranes might be safer, and have a better predictable resorption rate.The in vitro and in vivo studies of Won et al 37 showed a similar level of biocompatibility and bone regeneration potential of PLGA and collagen membranes (NB: 24.26 and 13.84%, respectively).The study asserted that PLGA membranes were more reliable in retaining form in the oral cavity than collagen membranes, which lost a tad of stability under wet conditions.Finally, the percentages of rate of rehabilitation of dehiscence defects for PLGA and collagen membranes were reported in the literature to be 70.20 and over 80%, respectively, whereby the complication rates were the highest for PLGA (37.4%) and lowest for the collagen membrane (10.4%). 31umming up all data from the present and previous studies, [31][32][33][34][35][36][37] apart from small differences, both types of membranes are biocompatible and suitable for GBR.Overall, BCPs have a great potential in dental treatments.To optimize the regenerative capabilities of bone, some studies led to promising results by adding bioactive agents, including silver nanoparticles, 38 advanced platelet-rich fibrin, 39 or mesenchymal stem cells. 40In future perspectives, more attention should be focused to hone the skills of synthetic BCPs in combination with tissue engineering and cell culture technology.

Conclusions
With the limitations of a clinical study in humans, the tested novel synthetic materials can be recommended for different dental surgical treatments, and may be an adequate alternative to autogenous bone.The present study reported an increased level of newly formed bone compared with current data from the literature.A statistically significant bone gain in length, width, and density in augmented sides could be observed after 8 months.It has been proven that both tested membranes have fulfilled all biological and mechanical functions for daily work.Further studies are necessary to verify the clinical effects of these biomaterials for long-term results as well as for patients with medical history or medical risks.

Ethical Approval
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Catholic University San Antonio of Murcia (protocol code CE061912 and date of approval: 07.06.2019).
Fig. 1 (A) Extractions of teeth 15 and 16 and immediate implant insertions in the upper law; (B) applied bone graft granules; (C) covered by PLGA membrane; and (D) result after 6 months of healing time.PLGA, poly(lactic-co-glycolic) acid.
Prospective Clinical Study with New Materials for Tissue Regeneration: A Study in Humans Kollek et al. 729 100%, embedded with methacrylate, polymerized, and sectioned using a diamond saw (Exakt, Apparatebau, Norderstedt, Hamburg, Germany).
5 software (Microms Image Processing Software, CID, Consulting Image Digital, Barcelona, Spain), and connected to a Sony DXC-151s 2/3-CCD RGB Color Video Camera.The areas of interest were marked, and their values were calculated digitally for the total percentage with the ImageJ software (W.Rasband, National Institutes of Health, Maryland, United States).The evaluation consisted of the measurements of new bone formation (NB), residual biomaterial (RB), and CT in relation to the total measurement area.Values were expressed in percentage.

Table 1
Radiographic measurements of total alveolar bone in cross-sectional view in CBCT and evaluation The biomaterials were well included in the host bone, marked by increased radiodensity around it.The mean bone gain in length, width, and density was 0.96 AE 3.33 mm, 1.22 AE 1.87 mm, and 387.47 AE 328.86 HU, respectively (►Table 2).Abbreviations: CBCT, cone-beam computed tomography; SD, standard deviation.a Statistical significance: p < 0.05.

Table 2
Mean bone gain in length, width, and density postoperative a Number of measurements.

Table 3
29west amount of NB (24.90%).Further studies compared the efficacy of BCP against autogenous bone and concluded higher results in NB with autografts: 38.63 to 41% 28 or 36.8%29versusNB in BCP of 26.68 to 33.70% 28 or 28.2%.
Abbreviations: CT, connective tissue; NB, new bone formation; RB, residual biomaterial; SD, standard deviation.the 22e et al22observed the first bone formation at around 12 weeks after implantation.BCP inhibited a too early osteoclastic resorption22; therefore, BCP seems to be more stable and predictable in behavior.In a systematic review, Troeltzsch et al 31 stated the clinical efficacy of grafting materials in alveolar ridge augmentations.After an investigation of a total of 184 papers, a horizontal mean bone gain of 2.2 AE 1.2 mm was described for synthetic biomaterials.The result is comparable to the mean width of 1.22 AE 1.87 mm of the present study.The highest vertical gain of 4.5 AE 1 mm was reached by autogenous bone mixed with allogenic or xenogeneic grafts, and the overall mean of all grafts was 3.7 AE 1.2 mm.