Alveolar ridge preservation with a collagen cone: Histological, histochemical, and immunohistochemical results of a randomized controlled clinical trial

Abstract Objectives The objective of the present study was to examine the influence of a combination material of a collagen cone and a collagen membrane on the healing process of extraction sockets with regard to histological, histochemical, and immunohistochemical parameters. Materials and methods In a prospective randomized clinical study, 10 patients (test group) received a collagen combination material after tooth removal. The extraction sockets of 10 other patients (control group) were left to heal without further intervention. Eleven ±1 weeks after tooth extraction, histological biopsies were performed in both groups at the time of implant placement. Subsequently, the biopsies were evaluated semiquantitatively in terms of histological, histochemical, and immunohistochemical parameters for the identification of factors of bone metabolism and vascularization. Results No significant difference between test and control group were found for any parameter. According to the descriptive data, the use of a collagen combination material seems to result in slightly higher values of the osteogenic Runt‐related transcription factor 2 (Runx2) and vascularization. Conclusion The histological, histochemical, and immunohistochemical analysis of ARP with a collagen cone combined with a collagen membrane showed no significant differences in terms of bone metabolism and vascularization.

The loss of bone volume can affect the surgical effort in terms of necessary grafting measures and consequently higher treatment costs.
To prevent bone loss, different actions after tooth extraction are taken to influence bone and soft tissue healing. Clinical concepts like the insertion of different bone substitutes, the closure of the empty socket with a membrane (Barone et al., 2008;Cardaropoli, Tamagnone, Roffredo, Gaveglio, & Cardaropoli, 2012;Ten Heggeler, Slot, & Van der Weijden, 2011), and the plastic covering with an advancement flap or a gingival transplant (Fickl et al., 2011) Bassir et al., 2018;Horowitz, Holtzclaw, & Rosen, 2012). Implants being placed in regenerated parts of the alveolar ridge show similar survival rates to implants being placed in natural bone, but it was not possible to demonstrate that one augmentation technique is superior compared to another technique based on implant survival rates (Chen et al., 2009;Corbella, Taschieri, Francetti, Weinstein, & del Fabbro, 2017). There was also no evidence for the superiority of one type of ARP intervention with regard to the formation of new bone (Corbella et al., 2017;MacBeth, Trullenque-Eriksson, Donos, & Mardas, 2017) or bone dimensional preservation and keratinized tissue dimensions (MacBeth et al., 2017).
The reduction of bone resorption with the use of a completely absorbable material is an innovative and promising concept. Extraction sockets that received a collagen cone combined with a collagen membrane after tooth removal in an animal study showed a significant reduction of bone resorption compared to extraction sockets without intervention (Kunert-Keil et al., 2015).
Parasorb Sombrero ® (Resorba, Nürnberg, Germany) is a new, completely resorbable material containing the combination of a collagen cone with equine collagen fibrils of Type I and a collagen membrane. To ensure simple and quick utilization, one product combines these two materials.
Up to now, there are no sufficient clinical trials on humans analyzing the material combination of a collagen cone with a collagen membrane (Annen, Schneider, & Schmidlin, 2014;Kunert-Keil et al., 2015).
In a recent clinical and histomorphometrical study, we have shown that there are no significant differences in terms of new bone formation and bone quality when performing ARP with Parasorb Sombrero ® after tooth removal compared to a control group without ARP (Schnutenhaus, Götz, Dreyhaupt, Rudolph, & Luthardt, 2018).
However, although histology and histomorphometry can indicate structural changes within the augmented socket, for example, osteogenesis or inflammation, the biological processes behind these changes remain elusive. Immunohistochemistry investigations allow to localize factors involved in these processes and to draw conclusions about the biological functions on cellular and even molecular levels.
Only a few immunohistochemical studies have been undertaken in biopsies from patients after augmentation of sockets. Most of them have focused on the remodeling of deproteinized bovine bone by using anabolic and catabolic bone markers (e.g., Milani, Dal Pozzo, Rasperini, Sforza, & Dellavia, 2016).
The aim of this study was to examine the influence of a combination material of a collagen cone and a collagen membrane on the healing process of extraction sockets with regard to histological, histochemical, and immunohistochemical parameters.

| MATERIALS AND METHODS
The study was conducted as a prospective controlled randomized clinical study according to the Declaration of Helsinki. The procedure and all the materials used were submitted to the relevant Ethics Committee of the University of Ulm and approved (No. 337/12, approved February 13, 2013).
The study participants were informed about the study before their participation, both orally and in writing, and gave their written informed consent. The study design corresponds to the previously published article of Schnutenhaus, Götz, et al. (Schnutenhaus, Götz, et al., 2018).

| Study population
Twenty patients with at least one tooth in the upper jaw that had to be removed and to be replaced by a fixed implant-supported restoration participated in the study. Two groups (test and control groups) of 10 patients each from a study with a total of 60 participants were included in consecutive order (Schnutenhaus et al., 2017).
Ten patients received ARP after tooth extraction (test group). In the other 10 patients, wound healing was allowed to proceed without further intervention. Inclusion and exclusion criteria are listed in detail in the previously published article of Schnutenhaus, Götz, et al. (Schnutenhaus, Götz, et al., 2018).
No differentiation was made in the indication of whether the tooth had to be extracted for periodontal reasons, carious destruction, or trauma, for example.

| Treatment protocol
The study took place in the private practice of the first author (S. I. S.), who exclusively performed all interventions and follow-ups. All participating patients were recruited in the same private practice in Hilzingen, Germany.
Interventions on the day of tooth extraction included local anaes- A nonsteroidal anti-inflammatory drug (600-mg ibuprofen) was prescribed.
Patients who needed a provisional removable restoration for either esthetic or functional reasons or at their personal request were given an interim prosthesis. Implant positions were determined by using an implant planning software (SMOP; Swissmeda, Zürich, Switzerland) and consequently transferred by means of a surgical template on the day of implantation which was 11 ± 1 weeks after tooth removal.
A trephine drill was used for sample collection at implant site.
The treatment protocol can be read in detail in the previously published article of Schnutenhaus, Götz, et al. (Schnutenhaus, Götz, et al., 2018).
Each sample was fixed by immersion in 4% buffered formaldehyde (Sörensen buffer) at room temperature (RT) for at least 1 day and subsequently decalcified for about 2 to 3 weeks in 4.1% disodium ethylene-diamino-tetraacetic acid-solution, which was changed every 24 hr. After hydration, tissues were dehydrated in an ascending series of ethanol and embedded in paraffin. Serial sagittal sections of 2-3 μm were cut, and representative slides were stained with haematoxylin-eosin, Masson-Goldner trichrome, and periodic acid-Schiff staining for histochemical detection of glycosaminoglycans and glycoproteins. In order to identify osteoclasts, selected tissue sections were stained to demonstrate tartrate-resistant acid phosphatase.

| Immunohistochemistry
Representative slides from the median parts of the sample series were deparaffinized, rehydrated, and rinsed for 10 min in tris-buffered saline. Endogenous peroxidase was blocked in a methanol/H 2 O 2 (Merck, Darmstadt, Germany) solution for 45 min in the dark. Sections were pretreated with phosphate-buffered saline containing 1% bovine serum albumin for 20 min at RT, digested with 0.4% pepsin for 10 min at 37 C, and afterwards incubated with the primary antibodies in a humid chamber. Antibody details and incubation protocols are listed in Table 1. Detection of antibody binding was performed with the peroxidase-conjugated EnVision ® antimouse system or the EnVision ® antirabbit/antigoat HRP-conjugated secondary antibodies (horseradish peroxidase, Dako, Glostrup, Denmark) diluted 1:50 and incubated for 30 min at RT. Peroxidase activity was visualized using diaminobenzidine (DAB) yielding a brown staining product, and slides were counterstained with Mayer's haematoxylin.

| Estimated sample size
Due to the lack of clinical data, no a priori sample size estimate could be obtained. The number of cases with 10 test and 10 control samples was based on the specifications of the ISO10993-6 (International Organization for Standardization, 2016). This study was therefore carried out as an exploratory study.

| Randomization
A randomization list was created for the overall study that included 60 patients (Institute of Epidemiology and Medical Biometry, University of Ulm, Germany). Assignment to the various groups was made in six layers. The data were stratified as follows: • By sex (two groups: male or female) • By region of the test tooth (three groups: anterior, premolar, and molar) T A B L E 1 Antibody details and incubation protocols The study director or a person authorized by him instructed the treatment center by fax as to the type of treatment to be performed according to the randomization list.

| Blinding
The laboratory received the samples in an anonymous form. The results were recorded on dedicated forms. The blinding was maintained until the samples had been completely prepared, analyzed, documented, and taken to a different place with a different operator than the researcher responsible for the histomorphological evaluation.

| Statistical analysis
For the metric target variables, the minimum, median, and maximum were reported. Nominal and ordinal features were described with their absolute and relative frequencies. Estimated sample size, randomization, blinding, and statistical analysis followed the same procedure as described in the previously published article of Schnutenhaus, Götz, et al. (Schnutenhaus, Götz, et al., 2018). in the control group, it was 48.8 (33.1-58.3) years. The randomized distribution of the teeth is shown in Table 2. Results of the study population have been previously published by the first author (Schnutenhaus, Götz, et al., 2018).

| Semiquantitative histological, histochemical, and immunohistochemical analysis
Besides the histomorphometric analysis, the 20 histological samples were histochemically and immunohistochemically evaluated for the identification of bone metabolism and vascularization factors.
The results are presented in Table 3. The Wilcoxon rank-sum test showed no significant difference between test and control groups for any parameter. According to the descriptive data, the use of a combination material seems to result in slightly higher values of Runx2 and VWF.

| DISCUSSION
The  protocol of delayed immediate implant placement, as prescribed for the present study. After 3 months, significant differences of the mineralization process are not to be expected due to the chronological sequence of the regenerating mechanisms (Trombelli et al., 2008).
Furthermore, a direct comparison with different studies is hardly possible because of high variation in treatment protocols and materials used as well as in histological analyzing methods applied for socket healing evaluation (MacBeth et al., 2017). It must be stated that many of the studies including histological examinations contain an insufficient number of patients and therefore should rather be considered case reports (Araujo & Lindhe, 2005;Engler-Hamm, Cheung, Yen, Stark, & Griffin, 2011;Kesmas et al., 2010). Another limitation is that the results of ARP measures are often not compared to unassisted socket healing (Checchi, Savarino, Montevecchi, Felice, & Checchi, 2011;Hoang & Mealey, 2012;Mardas, Chadha, & Donos, 2010;Margonar et al., 2012;Scheyer et al., 2016;Wood & Mealey, 2012). An evaluation of ARP at clinical and histomorphometric levels can take place if a control group with untreated extraction sockets is available along with the test group for comparative evaluation. Further on, the histologic results are influenced by patient selection. In patients who have a periodontal disease, new bone formation takes more time and is less predictable than in patients without a preexisting periodontitis (Ahn & Shin, 2008). The age of the patient is another influence on the healing process after tooth extraction, as angiogenesis and osteogenesis are delayed in aging patients (Nahles et al., 2013). In the present study, the patients of the control group were 4.5 years older than those of the test group. In a study of Brkovic et al. (Brkovic et al., 2012), immunohistochemistry was performed on five bone samples. Osteonectin, a phosphorylated, noncollagenous glycoprotein which is thought to be one regulator of bone metabolism, was detected in osteoblasts and osteoblast-like cells in two test groups, which included samples having been extracted at healed extraction sites 9 months after tooth removal. The authors assumed that remodeling of bone at the grafted sites was still ongoing (Brkovic et al., 2012). It has to be said that there was no control group for comparison without intervention after tooth extraction. The present study also showed the expression of proteins (Runx2, collagen, OC, and OP) in both groups. Immunoreactivity of OC, OP, and Runx2 was expressed slightly more pronounced in the test group, but no significant differences were found. Similar results were found in another study by Milani et al. (Milani et al., 2016), where higher expression of anabolic and catabolic bone markers was found in extraction sockets that were grafted with deproteinized bovine bone compared to sockets with no intervention.
According to the descriptive data, the use of a collagen combination material seems to result in slightly higher values of VWF. Biological processes during wound/socket healing could be positively influenced by the trend towards more vascularization with the use of such a collagen material. The collagen material used in this study had no negative impact on bone healing. There was no evidence of increased inflammation compared to the control group. The accumulation of an allogenic combination material consisting of calcium sulfate with a bovine bone substitute increased vital bone, probably because of a higher vascularization in the less dense material (Vance et al., 2004). The stimulation of angiogenesis by appropriate measures could support osteogenesis being dependent on sufficient vascularization.
Older patients with reduced angiogenesis might profit from the positive effect of ARP measures (Nahles et al., 2013).
However, with the methods employed in the present study, it can be concluded that the combination material has no proven influence or clinical relevance on the formation of new bone, neither can a possible influence be disproved. However, the clinical evaluation according to ARP showed a significant volume preservation in the buccal aspect of the alveolus with the collagen material used (Schnutenhaus, Doering, Dreyhaupt, Rudolph, & Luthardt, 2018). A potential advantage of delayed implant placement after a healing period of more than 3 months when performing ARP needs to be assessed in further investigations.
For studies with immunohistochemical targets, sample-size estimates might be of interest.