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Histological and Biological Response to Different Types of Biomaterials: A Narrative Single Research Center Experience over Three Decades

Background: In more than three decades of work of the Retrieval Bank of the Laboratory for Undemineralized Hard Tissue Histology of the University of Chieti-Pescara in Italy, many types of biomaterials were received and evaluated. The present retrospective review aimed to evaluate the histological and biological aspects of the evaluated bone substitute biomaterials. Methods: In the present study, the authors prepared a retrospective analysis after the screening of some databases (PubMed, Scopus, and EMBASE) to find papers published from the Retrieval Bank of the Laboratory for Undermineralized Hard Tissue Histology of the University of Chieti-Pescara analyzing only the papers dealing with bone substitute biomaterials and scaffolds, in the form of granules and block grafts, for bone regeneration procedures. Results: Fifty-two articles were found, including in vitro, in vivo, and clinical studies of different biomaterials. These articles were evaluated and organized in tables for a better understanding. Conclusions: Over three decades of studies have made it possible to assess the quality of many bone substitute biomaterials, helping to improve the physicochemical and biological properties of the biomaterials used in daily clinical practice.


Introduction
Studies related to bone substitute biomaterials derive from a necessity for biomaterials to help new bone formation, making it possible to reconstruct bone defects, while maintaining the biological and mechanical functions of the restored tissue [1][2][3]. Research on all biomaterials is necessary to ensure optimal results and the patients' safety [4][5][6].
Over more than three decades, many specimens of several types of biomaterials have been received and treated to obtain thin ground sections in the Retrieval Bank of the Laboratory for Undermineralized Hard Tissue Histology of the University of Chieti-Pescara in Italy. Histological and histo-morphometric analysis of the bone response with different grafts in different clinical situations associated to the in vitro response on cell cultures are certainly an important way to obtain information on the behavior of the various biomaterials, e.g., their different resorption patterns, bone formation with the use of particles or blocks, tissue response to the possible long-term persistence of some biomaterials. Besides light microscopy, other techniques can be used to evaluate histological slides containing biomaterials, i.e., Scanning Electron Microscopy, Transmission Electron Microscopy, Atomic Force Microscopy, Confocal Laser Scanning Microscopy, and Synchrotron Micro-CT [7][8][9][10][11][12]. These studies have helped in the evolution of bone substitute biomaterials, allowing reduction of morbidity due to the use of autogenous bone grafts, producing biomaterials with properties and physicochemical compositions similar to the host bone tissue. The present retrospective review aimed to evaluate the histological and biological results using different bone substitute biomaterials, in a time period of over three decades.

Materials and Methods
A retrospective evaluation of the scientific production of the Implant Retrieval Center Laboratory of University "G. D'Annunzio" of Chieti-Pescara in the last three decades was performed with databases PubMed, Scopus, and EMBASE in order to consider only the indexed scientific production of the Laboratory. The papers list has been obtained through the indexed papers lab archive.The articles screened were limited to papers dealing with bone substitute biomaterials for jawbone regeneration. The selected papers underwent a qualitative evaluation, analyzing the different biomaterials used, the study models, sample size, test and control group features, the study timepoints and the experimental findings.

Inclusion Criteria
Articles published up to January 2021 were included without language restriction. The articles screened were limited only to papers dealing with bone substitutes and scaffolds in the form of granules and block grafts for bone regeneration. The scientific articles included were verified for the qualitative analysis. According to the search criteria, human studies, in vitro studies, and animal model studies were evaluated. Articles that did not conform to the inclusion criteria and literature reviews were excluded from the review. The papers included were also categorized into block scaffolds, particulate graft and advanced experimental biomaterials.

Selection of the Studies
The experimental data and article selection were conducted independently by two expert reviewers (M.T. and A.P.). They used a particular designed data form by Excel software package (Office Microsoft, Redmond, WA, USA). Therefore, when the abstract was not available, the paper's full text was obtained and checked. Literature reviews, case reports, and book chapters were excluded from the qualitative analysis. For excluded articles, a description was performed of the reasons for exclusion ( Figure 1).

Results
A total of 86 papers were found and evaluated. Most of the available biomaterials in the past three decades in the market have been studied and were reported, i.e., anorganic bovine bone, equine bone, porcine bone, biphasic calcium-phosphate ceramics, phycogene hydroxyapatite, bio-glass, calcium carbonate, autologous bone, polylactide-polyglycolide, porous hydroxyapatite, beta-tricalcium-phosphate.

Anorganic Bovine Bone (ABB)
In most of the samples, the biomaterial grafted particles were surrounded by newlyformed bone. This newly-formed bone was in close and tight contact with the biomaterial particles' external surface, and no gaps, no fibrous, connective tissue, or foreign body reaction cells were found at the bone-biomaterial interface. In a few microscopic fields, osteoblasts were observed depositing osteoid matrix directly on the biomaterial surface, and, in other areas, a few osteoclasts could be observed at the interface with the grafted particles (Table 1) [13]. Slow resorption of the particles of ABB has been reported [13][14][15]. A study [16] found that it was possible to generate osteoclasts, starting from the mono-

Results
A total of 86 papers were found and evaluated. Most of the available biomaterials in the past three decades in the market have been studied and were reported, i.e., anorganic bovine bone, equine bone, porcine bone, biphasic calcium-phosphate ceramics, phycogene hydroxyapatite, bio-glass, calcium carbonate, autologous bone, polylactide-polyglycolide, porous hydroxyapatite, beta-tricalcium-phosphate.

Anorganic Bovine Bone (ABB)
In most of the samples, the biomaterial grafted particles were surrounded by newlyformed bone. This newly-formed bone was in close and tight contact with the biomaterial particles' external surface, and no gaps, no fibrous, connective tissue, or foreign body reaction cells were found at the bone-biomaterial interface. In a few microscopic fields, osteoblasts were observed depositing osteoid matrix directly on the biomaterial surface, and, in other areas, a few osteoclasts could be observed at the interface with the grafted particles (Table 1) [13]. Slow resorption of the particles of ABB has been reported [13][14][15]. A study [16] found that it was possible to generate osteoclasts, starting from the monocytes of peripheral blood, on the surface of slices of ABB, and that these osteoclasts were able to resorb the xenograft. ABB was a highly biocompatible and osteoconductive biomaterial with no foreign body reaction cells, no connective tissue, and no chronic inflammatory processes [14]. Some of the specimens containing ABB were retrieved, due to different causes, after many years [13,15,[17][18][19][20]. In all of these cases of long-term persistence of ABB in the tissues, lamellar, mature, compact bone was found at the bone-biomaterial interface, always in close contact with the particles, and, in some specimens under scanning electron microscopy, several projections of newly-formed bone were seen penetrating the ABB particles [17]. Moreover, relatively high concentrations of calcium and phosphorus found in the biomaterial particles decreased gradually toward the interface within the bone [17]. The residual grafted particles had not interfered with the formation of new bone in the site and had not produced any untoward or adverse effects. With the use of several biomaterials in sinus augmentation procedures, histology showed that in human biopsies retrieved after 6 months during implant insertion, the regenerated bone showed, in all cases, a similarity to D3 bone type, and only in a more extended period sample of ABB was the bone tissue comparable to D2 bone type, showing that, with the use of some biomaterials, an increase of bone density over time could occur [21]. Angiogenesis plays a relevant, pivotal role in osteogenesis, and a close temporal and spatial relationship between them has been reported [13,15,[17][18][19][20]. Angiogenesis can be evaluated by counting the number of newly-formed small blood vessels (micro-vessel density-MVD) and using immunohistochemistry, e.g., for Vascular Endothelial Growth Factor (VEGF). ABB seemed to be able to induce an increase in MVD that reached a higher value after 6 months (Table 1) [16]. A higher percentage of vessels and cells positive for VEGF were found in areas where there was newly-formed bone [21]. In a human study comparing autologous bone (AB) and ABB in sinus augmentation procedures, it was found that the difference in MVD and VEGF expression between sinuses augmented with AB and ABB was statistically significant, with higher values in AB specimens [19]. Similar results were found in another paper [16], with the highest values of MVD and VEGF expression in sites grafted with AB. In another human study on maxillary ridge defects, both sides augmented with AB and ABB presented a higher and statistically significant quantity of MVD compared to control, non-augmented sites [3]. Molecular studies found that ABB did not enhance the production of proinflammatory cytokines [21] and that the up-and down-regulation of several different genes could explain the reported bio-affinity of ABB for host tissues, its biological affinity to osteogenic cells, and its capability to stimulate osteogenic differentiation (Table 1) [21]. Bio-Oss particles did not interfere with bone-healing processes after sinus augmentation procedures and promoted new bone formation. This study can help clinicians to understand better the morphological characteristics of bone regeneration processes using Bio-Oss after 20 months and, most importantly, after a longer Under transmission electron microscopy, it was possible to characterize the bone-biomaterial interface; in the 20-month specimen an electron-dense layer was seen, whereas, almost no electron-dense lines were seen at the interface in the 7-year specimen.  [20] In this pilot controlled trial of the use of rhBMP-7, histological analyses showed that it resulted in the formation of less bone than treatment with inorganic bovine hydroxyapatite.
Histological and histo-morpho-metric analyses of biopsy specimens showed that there was significantly more new bone on the control side (19.9 (6.8)%) than on the test side (6.6 (4.8)%

Porcine Bone (PB)
Dual-phase porcine xenografts have different properties according to their composition and processing. Two different categories can be defined based on the varieties of bone present within the graft: 1.
collagenated cortical porcine bone Both families undergo a manufacturing process which preserves the main organic phase, represented by Collagen I protein, and prevents the ceramicization of the biomaterial which would limit the biological properties of the graft ( Table 2). Most studies performed on collagenated cortico-cancellous porcine bone found that grafted particles were surrounded by newly-formed bone starting as early as 3 months of healing [1,3,8,23,24]. Morphometric data, as extracted by histology and microCT analysis, conducted on post-extraction sockets, treated with collagenated cortico-cancellous heterologous pre-hydrated bone mix revealed a greater number of trabeculae filling the defect, compared to the spontaneously healed bone control samples, suggesting an improved strength of the socket, with histology showing the amount of biomaterial decreasing over time and replaced with newly formed bone. In contrast, less dense bone with wide marrow spaces was found in control samples. All data converge to confirm the good performance of collagenated cortico-cancellous porcine bone as substitute for the preservation of human maxillary ( Table 2) [8]. Clinical and histological outcomes indicated that collagenated cortico-cancellous porcine bone graft was found to be a highly biocompatible and osteo-conductive biomaterial that, thanks to its elevated interconnecting micro-porosity, could be used with success, alone or in association with autologous bone, in sinus augmentation procedures (Table 2) [23] A synchrotron study supports and validates the collagenated Cortico-Cancellous Porcine Bone graft capability of osteo-conduction, offering adequate support for tissue reconstruction, due to its biological characteristics and ability to support cell growth and differentiation [24]. In addition, the microCT analysis revealed a gradual decrease of the porcine graft biomaterial starting from the first week of culture, with the residual grafted particles not interfering with the formation of new bone in the site and without producing any untoward or adverse effects ( Table 2) [24].
An experimental study found that collagenated cortico-cancellous porcine bone granules embedded with growth factors (bFGF, VEGF etc.), derived from mesenchymal stem cells (MSCs) could promote an increase in new bone formation, in close and tight contact with the biomaterial particles' external surface, and stimulate vascularization in a rat calvarial defect model, without any inflammatory cell infiltration at the bone-biomaterial interface [25]. Collagenated cortico-cancellous porcine bone graft therefore can be considered a good reservoir for growth factor in a bioactive form allowing a good natural delivery system for bone healing. Finally, it was also found through an in vivo experiment that collagenated cortico-cancellous porcine bone mix and pre-hydrated CCCPB mix presented higher biocompatibility and were capable of inducing faster and greater bone formation compared to cancellous block of xenogenic bone [1]. On the other hand, collagenated cortical porcine bone showed no evidence of graft resorption after 4 months healing. The percentage of the residual graft material was the same after 4 and 6 months with no interference with bone regeneration processes and implant osseointegration. A slight increase in newly formed bone was found in the 6-month specimens (31%) as compared to the 4-month (28%) specimens [1]. Mature bone with many osteocytes was observed near the particles, and under Transmission Electron Microscopy all phases of bone formation (osteoid matrix, woven bone, and lamellar bone) were observed. All together these results suggest that collagenated cortical porcine bone substitutes, through their osteo-conductive potential, allow predictable placement of dental implants in the regenerated maxillary premolar and molar areas ( Table 2) [25]. The controlled release of active growth factors from porcine bone granules can enhance and promote bone regeneration.
The higher and lower intensities of vascular endothelial growth factor and NOS3 expression were prevalent in the sites grafted with autologous bone with significant differences with the controls (p < 0.05). In

Equine Bone (EQ)
Equine bone appeared to be a biocompatible biomaterial associated with new vessel ingrowth (Table 3). These small, newly-formed vessels are always found near and in close association with the advancing front of the new bone formation [26]. Higher intensity of VEGF expression was observed in newly-formed bone, whereas a low VEGF intensity was found in mature, compact, lamellar bone (Table 3) [26]. With the use of equine collagenated blocks, it was found that newly-formed bone was in close contact with the biomaterial [21,[28][29][30][31]]. An in vitro study, with the use of equine spongy bone slices, reported that osteoclasts could be produced from cells of the peripheral blood and that these cells were able to resorb the biomaterial (Table 3) [26].

Biphasic Calcium Phosphate (BCP)
Biphasic calcium phosphate (BCP) is an alloplastic biomaterial available in different microstructures, micro-and macro-porosities. The BCP particles showed a successful integration with the newly formed bone in mandibular sites [32] and in maxillary sinus augmentation procedures (Table 4) [3]. BCP could be adapted to large jaw defects through the CAD/CAM technique, and this biomaterial has shown a very good bone biocompatibility and osteo-conductivity [24,33]. In a study published many years ago, using a BCP composed of 50% hydroxyapatite and 50% beta-tricalcium-phosphate, it was found that many particles were surrounded by newly-formed bone and that some particles were undergoing resorption processes and were being gradually substituted by newly-formed bone [3]. With the use of BCPs with different percentages of the two constituents (Table 4) (HA and B-TCP), it was found that the particles were always surrounded by newly-formed bone (Table 4).

Calcium Carbonate
The particles were almost always surrounded by mature bone [35,36]. This biomaterial was clinically suitable for sinus augmentation procedures according to a successful new bone formation and graft integration (Table 5) [3,29,35]. The calcium carbonate-derived scaffold and graft could be obtained by coral aragonite or artificially sintered-procedure (Table 5) [3,35,36]. This biomaterial could be subjected to resorption with an higher efficacy then calcium-derived materials [3,35,36]. The graft porosity is able to promote the new bone formation in-growth and remodeling (Table 5)

Bioglass
Bio-glass was a highly osteoconductive material with the newly-formed bone around all particles, even those located in the central portion of the defects (Table 6) [2,38]. This biomaterial has resulted in being biocompatible and improved new bone formation in maxillary sinus lift [2]. The bio-glass bone substitutes are composed of minerals that are commonly present in the body, with calcium and phosphorous oxides proportions similar to the human bone percentage (Table 6) [39,40]. In literature, the bioglasses demonstrated an increase collagen depositions when in contact with the connective tissues [39]. Moreover, its porosity is able to increase the scaffold properties and the new bone formations when used to fill bone defects producing an in-growth of the osteoid matrix and the newly formed bone [41,42]. On the contrary, this biomaterial could be associated with a low fracture resistance and should be used in regions with no passive loading forces [41]. Different authors reported the antibacterial bio-glass's property when used for bone regeneration procedures (Table 6) [41].

Porous Hydroxyapatite (Porous HA)
Porous HA can be a suitable synthetic material for sinus augmentation procedures [43]. Biomaterial particles were observed in close and tight contact with mature, compact, and lamellar bone (Table 7) [21,34,[43][44][45][46]. A high quantity of newly-formed bone was found [43,47]. A large portion of the biomaterial particles was surrounded by bone [16,19,36,48,49]. Porous HA was reported to be of use also as joint prostheses [15,22,43,50,51]. The use of custom-made scaffolds made of porous HA Blocks has been reported that produced a vertical bone gain of 6.93 ± 0.23 mm after 6 months of healing (Table 8) [43,47].      Surg. 2013 [20] In this pilot controlled trial of the use of rhBMP-7, histological analyses showed that it resulted in the formation of less bone than treatment with inorganic bovine hydroxyapatite.
Histological and histo-morphometric analyses of biopsy specimens showed that there was significantly more new bone on the control side (19.9 (6.8)%) than on the test side (6.6 (4.8)%

Discussion
During all these years of research, different study models were used in our center. The evolution of the evaluation methods followed the progress of the techniques applied to determine the tested materials' biological quality. However, the methods most used in in vivo and clinical studies were histological and histomorphometric assessments of newly formed bone tissue. Large parts of these tested biomaterials have helped their implantation in the market or have evaluated those already available [3,23,62,63]. An important aspect is determined by the different origin of the xenogenic bone graft when used in bone regeneration procedure. Scarano et al. reported no significant differences between equine and porcine cortico/cancellous graft when used on standardized iliac defect [1]. Moreover, the authors reported a more highly significant new bone formation in grafted sites compared to the control empty bone defect [1]. The main characteristics observed, mainly in experimental studies, were not only the formation of bone tissue or the contact of the new tissue with the bone substitute but also the reabsorption of the material implanted in the cells present around the biomaterial (e.g., macrophages, giant cells multinucleate, osteoblasts, osteoclasts, and osteocytes). The impact produced by the material on the implanted tissue could identify the necessity for structural modifications (i.e., composition, granulation, and sintering) [43,64]. Thus, it is possible to improve the bone substitute for subsequent application in humans. In this long period, studies were made in granules and block formats, materials of different structures, but both of great clinical importance, mainly acting as a scaffold, the materials having osteo-conductivity as their main characteristic. Clinically, granules are most often used for small bone defects (e.g., dental socket), while blocks are reserved for larger areas (e.g., horizontal augmentation). The surgeon needs to take into account the structural and physicochemical characteristics of biomaterials. On the contrary, the majority of the evaluated graft biomaterials have shown a slow resorption, and the presence of residual grafted particles were found many years after the grafting procedure. [13][14][15]65,66]. This fact could be advantageous when the stability of the bone graft could be essential for the success of the regeneration, such as in sinus augmentation procedures (for helping in the contrast with repneumatization of the maxillary sinuses), in alveolar socket preservation techniques, and in severe mandibular atrophies [62,67,68]. Another advantageous effects is determined by the antibacterial role of some biomaterials and bioglasses, that could represent a useful strategy also for infected sites grafting in order to protect the healing phases of the bioscaffold osseointegration [69]. An in vitro study [70] found that it was possible to generate osteoclasts, starting from the monocytes of peripheral blood, on the surface of slices of ABB, and that these osteoclasts were able to resorb the xenograft. Many different advanced bioscaffold constructs have been studied such as graphene oxide-biomaterials, platelet derived growth factors/β-TCP constructs, interconnected porous hydroxy-apatite complex, rhBMP-7/deproteinised bone substitute, and autologous osteoblasts/polymeric scaffolds [20,28,45,[54][55][56][57][58][59][60]71]. Innovations correlated with new bone substitutes, such as rh-BMP and/or the incorporation of materials such as collagen, seeking improvement by bringing the ability of osteo-induction to improve the quality of the material presented, wer3 also studied, showing promising results, mainly the incorporation of collagen, which helps in the formation of the initial bone matrix and helps the arrival and adhesion of osteoprogenitor cells [24]. Concerning BMPs and mesenchymal cells, both are currently used in some countries in clinical procedures. However, it is possible to observe some studies that show limitations of these materials, either due to the exacerbation of bone tissue newly formed by BMPs or the formation of teratomas/hamartomas by mesenchymal cells in the region where these materials are implanted. More studies related to these materials are needed [24]. Among the studied materials, histological responses presented by the presented materials, mainly xenogenous and alloplastic, were excellent, considered safe materials, and capable of acting properly to reconstruct the new bone tissue [24]. However, they are matrices that will only assist in bone conduction. It is interesting to incorporate other components in these biomaterials, which may benefit the bone tissue into which they are implanted.

Conclusions
Currently, the search for biomaterials that will present properties similar to autogenous grafts is constant. The slow resorption rate of xeno-genic biomaterials could be useful when a higher bone graft stability is clinically advantageous for a successful dental implant positioning. After thirty years of research with bone substitutes, their safety and long-term effectiveness have been demonstrated. However, no biomaterial evaluated presented the same characteristics of the autologous bone. On the other hand, the use of xeno-genous or alloplastic grafts has been shown to be an excellent and safe option.  Informed Consent Statement: Not applicable.

Data Availability Statement:
All experimental data to support the findings of this study are available contacting the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.