Histomorphometric and immunohistochemical evaluation of collagen containing xenogeneic bone blocks used for lateral bone augmentation in staged implant placement

Different techniques and grafting materials have been used for the horizontal reconstruction of deficient alveolar processes before implant placement, resulting in different degrees of predictability and clinical outcomes [1]. Among the grafting materials, particulated xenogeneic materials have been extensively studied in both experimental and clinical studies and when combined with porcine-derived natural collagen membranes have resulted in predictable clinical and histological outcomes [2].This combined treatment has shown to be safe and efficacious in horizontal ridge augmentations resulting in regenerated bone with similar implant survival rates when compared to implants placed in pristine bone and demonstrating a low degree of morbidity and a low rate of post-operative complications [3, 4]. However, in clinical situations with severe bone resorption of the alveolar process, which results in non-self-containing bone defects, the use of particulate bone replacement grafts with its inherent weak volumetric stability may limit the predictability of the regenerative therapy [5, 6]. In these cases, dental implants are usually placed staged to the lateral bone augmentation procedure and autogenous bone blocks have been the state of the art procedure, resulting in superior results in mean horizontal bone gains when compared with guided bone regeneration with particulate bone grafts [4, 7, 8]. The use of bone blocks, however, has been associated with increased surgical time, morbidity [9, 10], and a higher frequency of post-operative complications [11, 12]. Moreover, the availability for harvesting intraoral bone blocks is limited [13, 14] and these autologous bone grafts may suffer a high degree of bone resorption during healing [15].

To overcome these limitations, the use of xenogeneic bone grafts as an alternative to autogenous bone blocks has been proposed [16]. Recently, a new equine-derived collagen containing xenogeneic bone block (CCXBB) was evaluated in preclinical studies [17, 18], demonstrating to be safe and adequate for ridge augmentation and better graft integration when compared to other xenogeneic bone blocks. Its performance in humans has been recently tested on 10 patients where these xenogeneic bone blocks were placed in single-tooth alveolar bone defects [19]. Clinically, a mean horizontal gains of 3.88 ± 1.75 mm was reported and the histological outcomes resulted in a homogeneous new bone formation within the CCXBB. These results were concordant with a recent histological study also reporting that equine bone grafts were biocompatible and underwent advanced remodelling at the time of implant placement [20]. Although this preliminary evidence on the performance and histological behaviour of equine bone blocks seems promising, there is still limited information when used in staged horizontal bone augmentation of large osseous defects. It was, therefore, the aim of this prospective study to evaluate the histological outcomes of CCXBB blocks used for lateral bone augmentation in large alveolar horizontal defects of at least two adjacent missing teeth.

Twenty-eight CCXBB blocks were placed in 15 patients that fulfilled the selection criteria (12 women and 3 men) with a mean age of 54.5 (SD 8.34).

Clinical results

The detailed clinical and radiographical outcomes have been reported previously [21]. In brief, one patient experienced pain and soft tissue dehiscence leading to removal of the graft material 3 days after the regenerative procedure. Another patient refused to proceed to implant placement after suffering an early dehiscence also leading to a complete removal of the graft. From the remaining 13 patients completing the study, the alveolar ridge width augmented from a mean 2.78 mm (SD 0.55) at baseline to 6.90 mm (SD 1.22) at re-entry, resulting in a statistical significant mean alveolar crest width gain of 4.12 mm (SD 1.32) Sixteen weeks after implant placement a second stage procedure and a soft tissue augmentation was performed (Fig. 7).

Although soft tissue dehiscence, with different degrees of graft exposure, occurred at different time points in 5 out of 15 patients (33.3%) (Fig. 8), 24 implants were placed in 13 patients. Table 1 depicts the data on survival rates at the time of loading. Three implants were lost in three patients at the time of abutment connection, and one patient presenting very narrow ridge at baseline (<2 mm) lost all the implants. Nevertheless, all implants could be replaced without additional grafting procedure.

Patient	Soft tissue dehiscence	Mineralized bone (%)	CCXBB (%)	Bone marrow (%)	Connective tissue (%)	Implant lost
1	No	22.56	25.26	15,443	36.03	No
2	Yes	0	28.49	0.000	71.50	Yes
3	No	30.39	21.10	14,248	33.24	No
4	No	31.59	21.79	13,982	32.62	No
5	No	41.44	11.90	17,259	25.88	Yes
6	Yes	39.41	5.88	54,461	0	No
7	No	24.31	13.21	32,813	26.84	No
8	Yes	26.19	21.09	52,714	0	No
9	No	23.39	30.18	39,455	6.96	No
10	No	17.73	15.79	26,590	39.88	Yes
11	Yes	12.95	27.09	10,104	48.83	Yes
12	No	37.45	21.37	35,035	3.89	No
13	No	42.31	28.35	29,129	0	No
%	Yes: 30.7 No: 69.2	26.90	20.89	26.24	25.05	Yes: 30.7 No: 69.2
Median		26.19	21.37	26.59	26.84
SD		12.21	7.35	16.43	22.07
IR		18.28	13.22	23.13	36.01
95% CI		19.52;34.28	16.44;25.33	16.31;36.18	11.71;38.39

Abbreviations: CCXBB Collagen containing xenogenic bovine bone, SD Standard deviation, IR Interquartile range, CI Confidence interval

The purpose of this investigation was to evaluate histologically and immunohistochemically the behavior of CCXBB blocks when used for staged lateral bone augmentation in severe human horizontal residual bone defects. Six months after the regenerative intervention using the CCXBB blocks, the mean increase in bone width was 4.12 mm and hence, this outcome allowed for the placement of dental implants in 11 out of 15 patients (73.3%). These results were concordant with the reported weighted mean width increases (3.90 mm (SD 0.38)) from a recent systematic review evaluating intraoral autogenous bone blocks [4]. These results were also similar to those reported with the use of allogeneic bone blocks (4.50 mm (SD 1.3)) [23] or with those from a pilot study using the same CCXBB xenogeneic bone blocks for the staged regeneration of single tooth bone defects (Schwarz, et al. 2016). In this study, in eight patients, the mean crestal width gain was 3.88 mm (SD 1.75) and implant placement was feasible in eight out of ten (80%) patients at re-entry [19].

To the best of our knowledge, the present investigation represents the first study reporting histomorphometric and immunohistochemical outcomes of the use of CCXBB for regenerating atrophic alveolar bone in humans. The healing after 26 weeks was characterized in most of the samples by newly formed mineralized vital bone containing viable osteocytes, as well as bone marrow and non-mineralized connective tissue. This new bone was observed in intimate contact with the residual CCXBB. The percentages of mineralized vital bone, bone marrow, and connective tissue were 26.9, 26.2 and 25.1%, respectively. Similar proportions have been reported with the use of allogeneic blocks [23, 24]. With autogenous bone blocks the relative tissue composition attained was 25.1% of vital bone, 18.1% of connective tissue, and 56.7% of necrotic bone [25]. Similarly, [26] reported 57.75% of non-vital bone when using autogenous bone blocks. In the present investigation CCXBB was present in 21.4% of the samples after 26 weeks of healing, what is in agreement with previous studies reporting histological outcomes of other xenogeneic bone replacement grafts placed for the regeneration of extraction sockets [27, 28]. In this indication, the percentage of residual graft was 39.8 and 33.4%, respectively.

When correlating the clinical results and the histological outcomes, there was a positive association between the presence of soft tissue dehiscence with CCXBB exposure and a diminished amount of new mineralized bone (p = 0.06). This lower amount of new bone within the xenogeneic graft suggests a lack of full graft integration and diminished vascular supply, what may have caused the soft tissues dehiscence. Similarly, the biopsies from patients who lost their implants had a statistical significant lower amount of MVB and a statistical significant larger proportion of connective tissue, what suggests that there is a direct relationship between the primary healing of the bone replacement graft, its integration with native bone and its healing to provide a biological base for dental implants to osseointegrate. These results corroborate the importance of minimal trauma during surgery, establishment of primary implant stability and avoidance of infection and micromotion during healing as key prerequisites for achieving dental implant osseointegration [29, 30]. In fact, the high incidence of early implant loss (29.2%) reported in this clinical study, is clearly higher when compared with epidemiological data from Sweden reporting early implant loss in 4.4% of patients and 1.4% of implants [31]. The delayed bone proliferative phase has also been described associated with other bone replacement grafts for bone regeneration [32] and with demineralized bovine bone mineral (DBBM) in the healing of fresh extraction sockets [33].

A high incidence of soft tissue dehiscence and implant failures has been reported in patients receiving fresh frozen allogeneic bone grafts for reconstructing severe alveolar atrophies (36.8% incidence of dehiscence and 31.5% incidence of implant loss) [34] and 21% of implant loss [35], respectively. With the use of a different equine bone block, a previous publication reported total removal of the graft in 50% of the patients and in 20% of them the implants failed [36]. The high incidence of soft tissue dehiscence occurring in this clinical study may also be explained by the extreme narrow crestal defects (mean crestal width of 2.78 mm) multiple teeth absence and non-containing defects, what needed in most of the cases to use more than one block graft. In fact, there was a positive correlation between the number of blocks used and the incidence of soft tissue dehiscences. The use of large grafts or more than one graft may have hindered an appropriate blood supply or colonization of the graft material with bone-forming cells [37].

The immune-histochemical results reported expression of osteopontin mainly at the border between mineralized vital bone (MVB) with CCXBB, what coincides with findings from previous reports [38–40]. Alkaline phosphatase (ALP) is considered as an early osteoblast differentiation marker [41]. ALP-positive cells were detectable, in all specimens on the periphery of MVB, associated to areas of new bone formation. These observations were also reported on a clinical study on guided bone regeneration (GBR) [41], as well as through the evaluation of the healing of particulate xenogeneic bone grafts (DBBM) [28]. Experimental research using immune-histochemical analysis for comparing early bone remodelling between autografts and allografts has reported comparable behavior for osteoprotegerin (OPG), alkaline phosphatase (ALP), collagen 1 (COLI), osteopontin (OPN), and osteocalcin (OSC), although an increased activity of tartrate-resistant acid phosphatase (TRAP) was seen in allogenic bone grafts [42]. In this investigation TRAP, which is a specific enzyme present in large quantities at the osteoclasts edge expressing bone resorption, was present in high proportions in all the analysed samples. On the other hand, OSC (bone matrix protein), predominantly synthesized by osteoblasts, has a fundamental role in bone formation (mineralization) and resorption [43]. Experimental studies have demonstrated the role of OSC during the early healing phases of osseointegration of dental implants [44]. In the present investigation, a statistical significant correlation between higher levels of OSC and implant loss was found. This association could be explained by a greater activity of bone modelling in these situations of deficient mineralization [45].

This prospective single-arm study has clear limitations to evaluate the efficacy of this bone regenerative intervention, since there is not a control group [46]. However, this investigation has shown excellent clinical performance and histological outcomes when CCXBB were used for lateral bone augmentation and when their integration occurred without soft tissue dehiscence.

Within the limitations of this clinical study, we may conclude that the use of CCXBB in combination with DBBM particles and a native bilayer collagen membrane for staged lateral bone augmentation in severe atrophic alveolar crests achieved significant horizontal crestal width allowing for staged implant placement in most of the patients. Histological analysis and implant survival records indicate that special attention must be paid to prevent soft tissue dehiscence.