The Role of Living, Resident Bacterial Biofilm Populations in the Osseointegration of Oral Implants

: This study presents a human jawbone model which explains bone health, disease and quality and the osseointegration interphase, at the cell level. Curation of 454 pyrosequencing data obtained from 153 bone, serous-effusion and granulation samples taken from 40 patients during two-stage dental implant installation, revealed a 20 phyla jawbone microbiome with total genera of 387. Multivariate statistical ordination of curated pyrosequencing data revealed six distinct ecologic groups in healthy and pathogenic ecosystems. Pyrosequencing of pristine bone (found in sites of congenital tooth absence) revealed a resident, health biofilm population, which now serves as an ecological health control and is a critical health reference in longitudinal multivariate statistical ordination of live biofilm, in population-shift. Osseointegration of a sterile, inert, commercially pure titanium surface which bio-mimics the surface and microarchitecture of pathogen-evading, smooth, uniform, healthy human cells, may define a persistent, beneficial association with symbiotic competence, where cooperative colonisation proceeds between beneficial bacteria, host stem-cells and biocompatible biomaterials, without immunomodulation. Dampening or restraint of the host immune response by beneficial bacterial sRNA is now reported in animal models and may be a key element in symbiotic homeostasis. We confirm that the clinical effect of surgical debridement and perfusion of an encapsulated, pathologic biofilm nidus to a vascularised health margin, beyond the confining sclerosis, is microbial population-shift. Population-shift can only occur in living biofilm communities. Enduring osseointegration can only be achieved by ensuring ecologic recovery of the bone bed to homeostatic health, that is, a bone bed that supports diverse, stable, resistant, resilient and even biofilm communities, with consortial regeneration of both healthy osseous internal and cortical architecture, before implant installation. The authors recognise that further research and replication studies of both pristine human jawbone and other bone qualities are required to provide additional knowledge and verification of our findings.


Introduction
Cellular Homeostasis and Pathologic Disturbance in Human Jawbone associations, including biotic and abiotic surfaces (Hentschel, 2021).The resident populations may be subjected to ecologic disturbance from tooth-borne infection and therefore present variable bone qualities, defined in microbial ecology across a clinical gradient of health and disease.Non-metric Multidimensional Scaling (NMDS) ordinated the curation of the pyrosequencing data into six distinct ecologic groups according to biofilm phenotype (Nelson, 2015), consistent with contemporary categorization of health and disease (Ciofu et al., 2022).In this way, health and disease may be defined at cell-level.
The Nelson and Viljoen bone quality index (Viljoen, 2019) describes five microbial ecologic categories across alternative stable ecologic states, which includes alternative stable pathologic ecologic states (Shade et al., 2012).This index describes two health states (H2 = health, higher bone density; H3 = health, lower bone density) and three disease states (D1 = sclerotic bone density, D4 = lytic bone density and D5 = mixed sclerotic/lytic bone density), where the potential for the regeneration of internal osseous architecture is defined by the variation in the site-specific resident biofilm phenotype.Both sclerotic bone (D1) and lytic bone (D4) are diseased bone characterized by disruption of normal trabeculation (Parfitt, 1962).Normal histologic variation is defined by Schroeder (1991), where the trabeculation is connected and varies only in coarse or fine trabecular mesh patterns and intra-trabecular density.The cortical envelope is of normal thickness without sclerotic densification.The D1, D4 and D5 biofilm disease states need to be bio-remediated before implant installation by Regenerative Surgical Debridement (RSD), to recover ecologic health and normal internal osseous architecture (now defined as H2 or H3), where ecologic diversity and stability supports symbiotic colonization of health microbiota on abiotic and biotic surfaces at the osseointegration interphase, without immunomodulation.
With factors like a sterile surgical field, clinical surgical competence and no intraprocedural or manufacturing contamination of the implant surface, the outcome at the osseointegration interphase will be delivered by the ecologic health/disease status in the bone bed and the resident, live biofilm phenotype in the bone bed (Ciofu et al., 2022).Furthermore, the surface topography and microarchitecture of the implant surface (Sa value, or surface roughness) should biomimic the osseous ground substance (Brånemark et al., 1985).Percival et al. (2015) describes the smooth, uniform surface of healthy human cells as "pathogen-evading", while Mukherjee and Bassler (2019) describes such (smooth) surfaces as "inhibiting cell-to-cell communication quorum sensing" by retaining microbial exposure to cell (or implant) surface micro-fluidity which dilutes and clears pheromone autoinducer cells, keeping their numbers below a threshold level.If micro-fluidity is disrupted by a rough surface topography or microgrooves, this may reactivate pathologic persister-cell dormancy and phenotypic relapse infection (Lewis, 2010, Fisher et al., 2017).The key issue is that the microbes of interest are 1-2 microns, so an interpillar surface separation of less than 1 micron (Sa<1µ) will prevent bacteria slumping and adhesion onto surface recesses (Graham and Cady, 2014), thereby retaining exposure to cell surface micro-fluidity, with concomitant inhibition of cell-to-cell communication via quorum sensing (Mukherjee and Bassler, 2019).
In contrast to a sterile bone model, where the principal issue is the upregulation of pluripotent mesenchymal stem cells in response to implant installation (note that this model gives no consideration to competition from resident bacterial bone "guests", in a "race for the implant surface") (Gristina et al., 1988), the ubiquitous biofilm bone model attaches great import to surface roughness, because of the increased adhesion of bacterial biomass (including pathogens).Where the microbes of interest are 1-2 microns (Graham and Cady, 2014), roughened surfaces with an interpillar distance of more than 1 micron significantly increased adhesion of bacterial biomass and pathogens (Bermejo et al., 2019), with both increased surface area for adhesion (Berne et al., 2018) and facilitated activation of quorum sensing and ultimately, endotoxin production.Costerton et al. (1978) stated that adhesion plays "the central role in the success of pathogenic bacteria.Prevention of adhesion is an effective way to combat or prevent bacterial infection".
An abiotic implant surface that is rougher than the biotic surface may create preferential microbial adhesion with potential loss of genera that contribute to the resilient homeostasis of the resident biofilm (Ehrlich et al., 2008).Thus, a hard-won population-shift, recovered to ecologic homeostasis, by what may have been repetitious surgical debridement, may be destabilized by genotypic response to the surface, with the loss of constituent genera, creating a potential impediment to symbiotic, cooperative, homeostatic colonization at the osseointegration interphase.Pathogenicity may re-emerge in populations that are missing normal constituents and ultimately, there is agreement in both theory and experimentation in macroecology that "the removal or addition of even a single species can cause drastic community changes", leaving simplified, lower diversity communities, more susceptible to invasion and disease (McCann, 2000).Costerton et al. (2003) suggested that "a resident biofilm population may preclude colonisation by pathogens", maintaining cellular homeostasis by avoiding pathways of pathologic capillary lysis, which includes direct endotoxin lysis from gram negative pathogens (Brånemark and Urbasschek, 1967) and compression lysis from inflammation and raised intraosseous pressure (Wannfors and Hammarström, 1989;Wannfors and Gazelius, 1991).Cellular health and normalized function rely on normalized resident microbiota (Van Oppen and Blackall, 2019).Lasting ecologic diversity in beneficial resident microbial symbiosis underlies "mechanisms of health, resilience and cellular homeostasis" (Visick et al., 2021).
Interestingly, Visick et al. (2021) state that we now recognize that in resident biofilm populations there are "a much greater number and greater complexity of beneficial microbes in our bodies", compared to pathogens, where normal resident, beneficial, live biofilm communities sustain biologic health defined in ecologic and histologic cellular homeostasis (Van Oppen and Blackall, 2019).The superior numbers of beneficial bacteria, coupled with the smooth and uniform machined implant surface and its propensity to evade pathogen adhesion and thereby inhibit quorum sensing, provides the platform for the enduring success of osseointegration.
Supporting this is evidence from an animal model that some bacterial sRNA was able to dampen and restrain the immune response (Moriano-Gutierrez et al., 2020) and hence reduce the inflammatory disturbance to a shortterm pulse disturbance without transition to a less stable, lower diversity alternative ecological state (Shade et al., 2012).This may play a key role in sustaining a symbiotic, cooperative colonization of diverse beneficial health microbiota on abiotic and biotic surfaces at the osseointegration interphase, progressing a passive, enduring, osteoblastic anchorage without an immunobiological event, immunomodulation or foreignbody response.This results in a lasting symbiosis between mammalian host cells and their supported, healthy microbiota (Visick et al., 2021).Weinstein and Darouiche (2001) observed that the ubiquitous nature of biofilm meant that we could circumvent pathologic biofilm to either less pathogenic or nonpathogenic biofilm, but not remove it.Costerton (2007) described a disturbance to a resident biofilm population which resulted in the complete or partial replacement of that population by a more disease orientated population as "population-shift".Bioremediation and circumvention of pathologic residency before implant installation could be achieved by regenerative surgical debridement to a vascularized margin, through the sclerotic encapsulation.The clinical effect of the debridement was population-shift.The health ecology of a future implant bone site could be recovered with extinctions and additions of phyla, according to habitat fitness, via a microbial convergence of the reconnected, adjacent health microbiota (Bartow-McKenney et al., 2018).
We hypothesize that enduring cellular homeostasis in jawbone supporting osseointegration requires a stable vascular nutrition, with a connected, healthy capillary microvasculature, a stem cell rich periosteum, supporting histologically stable microenvironments with connected trabeculation which sustains beneficial, ecologically diverse, resident biofilm populations, which are both resistant and resilient to pathogen invasion.This is achieved by passive osteoblastic anchorage and symbiotic, cooperative colonization of health microbiota on inert abiotic and biotic surfaces, without immunomodulation.Biocompatibility requires biofilm compatibility.
As Botton et al. (2006) stated "It is time therefore to revisit the existing concepts and hypotheses and discuss their applicability to ecosystems ruled by microbial processes".

Patient Selection
Forty patients requiring osseointegrated dental implant therapy were prospectively enrolled into the study as they presented.All surgical implant sites were twostage, full-flap, sterile surgical procedures, ad modum Brånemark (Adell et al., 1981).Ethical approval was obtained from the Human Research Ethics Committee, The University of Sydney (HREC reference number 07-2007/9962).Patients were issued with a patient information statement and gave written informed consent.

Sampling
Multiple samples were taken from the same site to increase the probability of sequencing the entire ecological community.Samples were obtained from osteotomies in all four dental quadrants, within postextraction regenerated or regenerating jawbone and were defined as: a) Inherited Apparently Healed Bone (IAHB) samples taken from an apparently healed edentulous site where extraction of the infected tooth had been done previously in another dental practice (n = 29).The extraction protocol had conformed to current teaching where the diseased tooth was extracted without socket intervention, with the expectation of spontaneous bony healing and preservation of bone sterility b) Debrided Apparently Healed Bone (DAHB) samples taken from an osteotomy prepared in regenerated bone which had apparently healed for at least 3-6 months after a surgical debridement of an infected tooth socket beyond sclerotic confinement or surgical debridement of osteolytic/osteosclerotic "apparently healed" edentulous bone to an ecological health margin (n = 76) c) Congenital Absence Bone (CAB) osteotomy samples were taken from two patients presenting disease-free bone sites with congenital absence of upper lateral incisor teeth (n = 9).These sites had never supported a tooth and were thus deemed to contain disease-free bone.One site also had both deciduous and permanent congenital tooth absence

Sample Collection
Blood and tissue fluid samples were taken by paper point effusion, using sterile size 80 Maillefer endodontic paper points.Two sterile paper points were held in place in the sample site for a minimum of 15 sec and transferred to vials containing Reduced Transport Fluid (RTF) with 30% glycerol and frozen.Where concern existed for dilution of the sample by a transport fluid medium, paper points were placed in dry sterile vials and frozen.Bone samples were retained on the drill flutes and placed in the vials and frozen.
Samples included: Bone retrieved from the flutes of round surgical guide drill • Bone retrieved from sterile surgical bone filter • Bone retrieved from infrabony lesions • Bone retrieved by surgical excision using rongeurs Samples were then transported to Macquarie University, Sydney, Australia, extracted and subjected to PCR amplification using the 27F 16s rRNA eubacterial universal primers.Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA).Bacterial Tag-Encoded FLX Amplicon Pyrosequencing (bTEFAP) of the V1-V3 regions of the 16S rRNA gene was commercially performed by Molecular Research Laboratory USA using the Titanium platform (Roche).This bTEFAP method has been described by Dowd et al. (2008) and utilized in characterizing a wide range of environmental and health-related microbiomes.

Scanning Electron Microscopy
During our initial research, SEM was carried out on multiple samples to confirm that we were dealing with living bacteria in biofilm communities and not planktonic bacteria, or dead bacterial remnants.This is discussed further in discussion.
The samples were fixed in 3% glutaraldehyde for 24 h, dehydrated through increasing concentrations of ethanol and immersed in hexamethyldisilane (Sigma-Aldrich) 50% for 10 min and 100% for 310 min, before being aspirated and evaporated dry overnight.They were mounted on metal stubs and sputter coated with 20 nm gold film.The samples were imaged in a JEOL 6480 LV scanning electron microscope (Japan electron optics Laboratory, Japan) with a voltage of 10 kV and a viewing distance of 20 mm.

Statistical Methods
Many variables and data sets with up to 94% zero cells were detected.As shown in results the need for nonparametric analysis of the groups dictated the use of: a) Univariate Shannon-Weiner index of alpha diversity together with the jack-knife standard error b) Multivariate analysis by ordination with Non-Metric Multidimensional Scaling (NMDS) c) Analysis of Similarities (ANOSIM) was used to test the significance of a difference between groups.Similarity Percentage (SIMPER) was used to define the compositional differences within and between the groups Non-constrained non-parametric multidimensional scaling analysis in 3D were presented for all ordinations.Many runs of ordination were performed with bray curtis similarity measure and the Sørenson Incidence-based Index to achieve the method with the least stress value.This method of NMDS analyses the matrix of pair-wise similarities between samples in the data set.Similarity or dissimilarity can be measured.Analysis of similarities using bray-curtis similarity index was chosen to do this as it uses quantitative data and not just presence or absence of information.If assigned groups such as IAHB or DAHB are meaningful, samples within groups should be more similar in composition than samples from different groups (pisces conservation).
An ANOSIM significance test ranks similarity within and between groups and compares this with similarity generated by random chance.A square root transformation is chosen to normalise non-parametric data.

Contamination
Contamination of bone samples containing exogenous microbes has been problematic in previous endodontic studies (Siqueira and Lopes, 2001).Therefore, to detect for contamination, at the time of bone sampling, additional samples were taken from saliva and plaque and the microbial assemblages compared between sites using Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis of the 16S rRNA gene.A 918bp gene fragment of 16S ribosomal RNA was amplified using bacterial universal primer 6-FAM fluorescent dye labelled primer 8F (5'-Fam-AGAGTTTGATCCTGGCTCAG-3') and 926R (5'-CCGTCAATTCMTTTGAGTTT-3').The 25 µL reaction mix contained 1X PCR buffer, 2.5 mm MgCl2, 200 µm dNTP, 400 nm forward and reverse primer, 1U Immolase Polymerase (Bioline) and 100 ng DNA template and was subjected to 95°C for 10 min, denaturation at 95°C for 15 sec, annealing at 56°C for 45 sec and extension at 72°C for 2 min and a final extension step of 72°C for 10 min.The PCR products were purified using QIA quick PCR purification kit (Qiagen) and digested with 20U restriction enzyme HhaI (New England Biolab) at 37°C for 3 h.The fluorescent labelled terminal restriction fragments were separated by capillary electrophoresis using ABI 3130 genetic analyser and TRFLP profiles were analysed by comparison of abundance peaks.

Curation of Pyrosequencing Results
Using DNA (culture-independent) analysis, all 153 clinical samples, including the two CAB samples, were positive for the presence of bacterial biofilm.The 20 phyla found in human jawbone and the number of samples with bacteria in each phylum is shown in Table 1.All implants were installed as two-stage procedures.
Surgical debridement protocols were developed over time (DAHB) meaning that some osteotomies preceded the introduction of this protocol or presented as nondebrided Apparently Healed Bone sites (IAHB).
To provide contemporary DNA-based verification of the sampling integrity, bone samples were accompanied by samples of the potential contaminants of plaque and saliva, taken at the time of the surgery for analysis of each of the three samples by TRFLP.This analysis is based on variation in the 16S rRNA DNA-fingerprinting technique to reveal community abundance peaks.In every sampling site, site-samples had different microbial assemblages and it could be concluded that each of the three samples were uncontaminated by the other.
Examples of TRFLP graphs, generated from samples taken at the time of osteotomy preparation and sampling and including saliva and plaque, are shown in Figs.4b-d.

NMDS of Curated Pyrosequencing
NMDS analysis provided multivariate ordination (pisces conservation) of the curated 454 pyrosequencing of all clinical samples, in 3D.NMDS places the most ecologically similar samples closest together and the least similar further apart with the best arrangement in a small number of dimensions determined by a parameter termed the "stress".The best ordination will be the one with the lowest stress.Several ordinations were tried and compared with different starting positions, as recommended (Henderson and Seaby, 2014).Multivariate analysis is highly site-specific as it detects the effect of disturbances on biodiversity.NMDS is the multivariate approach most suited to data in disturbance studies (Magurran and McGill, 2011).
Communities can be tracked through disturbance and the recovery trajectory, separated by ecological similarity or dissimilarity against CAB ecological health control.NMDS with the bray curtis (pisces conservation) similarity measure, revealed a clear separation of six ecologically distinct groups in the sample data with a good representation in reduced dimensions (3D stress = 0.17) (Fig. 1).
A total of 387 genera were identified across all samples.Surgically Debrided groups (DAHB) pooled microbiome results contain all of the 51 genera found in the health control (CAB) (Fig. 2), although across a clinical gradient of health and disease it was clear that some debrided sites achieved only partial ecological health recovery (DAHB Group 1) and required further intervention (i.e., more than one debridement) to create a 'cleaner' ecological recovery closer in ordination to the health control before implant installation.Non-debrided ordinated groups (IAHB) had significantly different genera in their compositional structure to the health controls.These groups ordinated closer to disease and biomaterial stability in such bone beds may be challenged by lower diversity and ambiguous ecological stability.The soft bone category is identified by NMDS analysis as a high pathogen subgroup, within nondebrided IAHB (Fig. 1).

Analysis of Similarity (ANOSIM). Statistical Significance Between CLINICAL Groups
The results of an ANOSIM between the six ecologically distinct groups are shown in Table 2.
All pairwise comparisons were statistically significant (p≤0.05)except for IAHB healed extraction site verses IAHB soft bone (p = 0.996).An ANOSIM significance test, ranks similarity within and between groups and compares this with similarity generated by random chance.A square root transformation is chosen to normalise non-parametric data.ANOSIM tests whether the effects of disturbances on group compositional community structure are statistically significant (Magurran and McGill, 2011).Despite the ecological closeness of IAHB groups revealed in the NMDS ordination, a Similarity Percentages analysis (SIMPER) of the sample groups revealed significantly higher levels of known pathogens present in the IAHB soft bone group than in the IAHB Healed extraction site group (IAHB soft bone 89% versus IAHB healed extraction sites 75%).The osteolytic presentation of IAHB soft bone may be explained by its microbiological compositional structure.No osteosclerotic or soft osteolytic bone qualities were reported in DAHB samples.

Ecological Health Control
DNA molecular 454 pyrosequencing of bone samples from healthy sites of congenital tooth absence in the adult human jaw would appear to be without precedent (Fig. 2a).
All sample sites (n = 9) were taken from two healthy adults in disease-free mouths.Both were non-smokers with no compromising comorbidities and both were positive for the presence of biofilm communities in the osteotomy sites (Fig. 2b).One site in the position of the upper permanent lateral incisor had congenital absence of both deciduous and permanent teeth.The results of CAB curation contradict the presumed sterility of human jawbone bone and is considered evidence that even healthy jawbone harbours a (beneficial) resident biofilm population.These populations define a homeostatic health control reference for jawbone as an ecological health control (Fig. 2b) in microbial compositional structure at the level of phyla and genera.
The identification of resident biofilm communities in sites of congenital adult tooth absence represent control sample groups, to be compared with continued NMDS ordination of disturbed communities, in order to monitor their recovery and health status prior to implant installation.In this way, it may be possible to develop surgical debridement methodologies adapted from orthopaedic experience with chronic osteomyelitis.This ultimately may be a benchmark for multivariate analysis to identify alternative stable states that may favour improved clinical outcomes in jawbone osseointegration.
The outcome of two implants in CAB2 bone (healthy/pristine) is seen in Fig. 3 after 6 years of function.
Variable bone quality could now be defined as a microbial health/disease phenomenon across a spectrum of non-pathologic to pathologic microbial community assemblages.Pooled results of ecological similarity revealed that only surgically debrided sites and saliva control sites had all 51 genera of the health control (Fig. 4e).Site specific bone quality reflected site-specific dominance of beneficial health or pathogens in microbial community structures (Kolenbrander, 2011).Absence of trabeculation meant that regeneration had been impaired and osteosclerosis was associated with all chronic bone pathology (Parfitt, 1962).The category most ecologically dissimilar to homeostasis was the failed implant (Fig. 4e).
The primary contribution of bone bed dynamics to implant outcomes is to restore heath ecology, which may now be referenced against the ecological health control of pristine bone of Congenital tooth Absence (CAB).
Dental implant failure as a dual biofilm infection of both the implant surface and chronic osteomyelitic infection of bone (Zimmerli, 2014) achieved the most significant ecological pathogenicity with 39 genera, distinct from the 51 genera of the health-control, even more significant in pathogenicity than the chronic periapical granuloma (disease control), with 30 distinct genera, which had previously been described as a sterile inflammatory process.

Comparison of the Exponential H (Alpha Diversity), CAB Against IAHB and DAHB
Constituent pooled phyla, genera, species or Operational Taxonomic Units (OTUs) were examined using Shannon Wiener exponential H alpha-diversity (Pisces Conservation) for richness and evenness in various clinical states and sample types.Exponential H Shannon Wiener diversity for distinct clinical groups, shows the CAB health-controls with the highest pooled alpha diversity (exponential H).There is a maximised diversity disparity between the failed implant and the diversity of the ecological health-control; the former is 3.9 times lower in diversity.Botton et al. (2006) noted that ecologic diversity and stability are almost inseparable ecological concepts.
A comparison of the alpha diversity was also made between the CAB control and both DAHB and IAHB.DAHB (partially or fully ecologically recovered bone) was more diverse than IAHB (Table 3).

Congenital Absence Bone (CAB), Dissection into Two Groups
The initial model of congenital absence bone was dissected into 2 groups, control CAB1 and control CAB2.Ordinal separation was confirmed by a significant difference in microbial assemblage composition (Fig. 5).This may imply that even in pristine/healthy bone, the microbial community assemblages may be unique for each patient.
Both members of CAB groups were designated as controls, since the microbial assemblages of each were situated in clinically pristine, non-diseased bone.

NMDS Validation of Health Controls
NMDS ordination allows us to visualise populationshift in the bone bed following RSD and also validates the health controls CAB1 and CAB2 as valid reference points of bone-bed microbial health.If RSD resulted in a population-shift away from our two health controls, then they may not be representative of bone health/homeostasis (Fig. 6).

Visualization of Longitudinal Population-shift in Same-Site Osteotomy Following Implant Failure in Soft Bone (D4), Followed by RSD and Successful Revision Using Pie-Charts
Figure 7 shows three pie charts constructed from pyrosequencing of same-site constituent genera (simper analysis) in Sample 54F (soft bone-D4), sample 56E (implant failure-D4) and sample 91E (post-debridement health recovery-H3), which correlates with improved bone quality and recovery of ecologic diversity, richness and evenness in genera, as determined from longitudinal univariate exponential H alpha diversity statistical analysis.

Using Exponential H Alpha Tabulation
Analysis of a longitudinal series of samples, taken at the initial implant placement (54F), then later at the time of implant failure (56E) and then again after RSD (91E) is shown in Table 4.The initial bone site supported 6 phyla and 18 genera (alpha H 7.9) and alpha H dropped to 1.147 at the time of implant failure with just 3 phyla and 9 genera.After successful RSD, the bone bed was found to support 11 phyla and 53 genera, with an alpha H of 27.8.The revision implant successfully Osseo integrated in the ecologically recovered bone bed.

Using Shannon-Wiener Exponential Diversity Analysis
Recovery of ecological diversity within the implant bone bed can be shown using Shannon-Wiener exponential diversity analysis (Fig. 8).

Example of the Clinical Effect of RSD on Population Shift in Debrided (Gp2) Versus Inherited, Non-Debrided Bone
NMDS maps live biofilm extinctions and additions according to habitat fitness, revealing the clinical effect of surgical debridement as population-shift (Fig. 9).Multivariate analysis is highly site specific and therefore highly relevant to mapping disturbed communities through the recovery trajectory, over time (Warwick and Clarke, 1993).
It considers the identity of species, rather than richness or diversity (Hewitt et al., 2005).NMDS may be the most appropriate multivariate approach for data retrieved from disturbance studies (Magurran and McGill, 2011).

Example of Longitudinal Same-Site Population-Shift Before Implant Installation
Immediate implant placement (that is, at the time of tooth removal) has become an accepted protocol, however, the validity of this protocol may be questionable, as our NMDS ordination exposes the risk of this protocol in placing the implant into a diseased bone bed. Figure 10 shows ecologic recovery of a potential implant site before implant installation.The patient presented with a fractured post-core and crown and therefore the site was deemed unsuitable for immediate implant placement.The bone-bed was diagnosed as D5 (osteolytic/sclerotic) and was debrided at the time of tooth removal, in preparation of implant placement.(Viljoen, 2019) anatomy, correlating with pre-existing pathology of long duration and lower diversity.When both genera were curated in high abundance from the same site, Streptococcus also presented typically with high "species" (Operational Taxonomic Units) numbers and the sites were surgically difficult to recover ecologically, often requiring multiple debridements, especially in the posterior mandible and sites in the anterior maxilla with diffuse "bicortical" sclerosis.

NMDS of Health Biomarker Rheinheimera
In the curation of natural and pathogenic ecosystems, the genus Rheinheimera (Fig. 11) proved to be a predictive health biomarker in constituent genera, while Streptococcus (Fig. 12) and Propionibacterium proved to be predictive pathologic biomarkers, when in significant abundance in the community profile.NMDS ordination of the health biomarker Rheinheimera was undertaken of four bone groups including control CAB1, DAHB Gp2, IAHB (healed extraction site) and IAHB (soft bone).Note how DAHB Gp2 ordinated closer to the control CAB1 than the two non-debrided groups of IAHB (Fig. 11).

NMDS of Disease Biomarker Streptococcus
NMDS ordination of the disease biomarker Streptococcus was undertaken of four bone groups including control CAB1, DAHB Gp2, IAHB (healed extraction site) and IAHB (soft bone).Note how the two non-debrided groups of IAHB ordinate closer to each other and away from the health control CAB1 and the ecologically recovered DAHB Gp2 (Fig. 12).

Does Human Jawbone Support Living Bacterial Biofilm Communities?
454 pyrosequencing is a method suited to the detection of bacteria living in biofilm communities, whereas culture techniques will only detect bacteria living as "freefloaters" or planktons (Clarridge III, 2004, Dowd et al., 2008;Wolcott et al., 2010;Tuttle et al., 2011;Ehrlich and Arciola, 2012;Rhoads et al., 2012;Tipton et al., 2017).Site-specific pyrosequencing revealed an almost infinite number of alternative ecological states, in both healthy and pathogenic ecosystems (Shade et al., 2012;Weinstein and Darouiche, 2001).This strongly suggests the routine presence of bacterial biofilm populations in the jawbone.454 pyrosequencing does not distinguish between the DNA of living bacteria and dead bacteria and therefore, it could be argued that the presence of bacterial DNA in human jawbone is an end-product of the actions of the innate immune system.We have however, shown convincingly that, using orthopaedic-derived debridement techniques which link healthy tissue with niduses of diseased tissue, we can cause a bacterial population-shift, back to a health population, which ordinates close to our health controls.Only living bacterial biofilm communities can be population-shifted.Since RSDs can result in a population-shift that ordinates closer to our health controls, this suggests that our health controls too are living microbiome communities (Fig. 6).
Figure 13 provides visual (SEM) confirmation of a longitudinal population shift from rods to cocci, following debridement and 3 months of healing.Figure 14 is an example of radiographic, post-RSD healing at 3 months, following 10 years of persistent osteolytic/osteosclerotic (D5) bone histopathology.

Curation
Curation of our data found polymicrobial biofilm assemblages in 100% of jawbone samples.In all sites in the dental implant treatment sequence, starting with the extraction socket and including granuloma and congenital absence, we found polymicrobial biofilm assemblages, both in health and disease.To reduce bias in pyrosequencing sampling, multiple serous effusion, granulation and bone samples were retrieved from each site.One of the principal variations was to sample at different depths and at different widths within the jawbone.The pyrosequencing may suggest the existence of an infinite number of alternative stable states as microniches in both healthy and pathogenic ecosystems.The results indicate that considerable site specificity exists for each biofilm assemblage.

Ordination
Ordination is a collective term for multivariate techniques which summarize a multidimensional dataset in such a way that when it is projected onto a low dimensional space, any intrinsic pattern the data may possess becomes apparent upon visual inspection (Pielou, 1984).Multi-variate analysis of the pyrosequencing results of our identified clinical groups reveals a progressive reduction in known pathogens as the sites reduce their ecological distance, in ordination, relative to the health controls.
It is apparent from the NMDS ordination that surgical debridement produces a wound with reduced levels of pathogens and is ecologically closer to the compositional structure of health microbiota in CAB1 and CAB2, with increased community diversity (Fig. 6).It is suggested that the ecological distance between DAHB Group 1 and DAHB Group 2 results from the fact that the two debrided groups represent alternative stable states, but in different phases of ecological recovery.The percentage of known pathogens retained in the compositional structure of pooled results of DAHB Gp1 may suggest that a repeated debridement would result in an ecological outcome which would ordinate closer to DAHB Gp2.In the transitional gradient between health and disease DAHB Gp2 is less pathogenic versus DAHB Gp1 and its ordination reflects this in its closeness to control CAB1.
Of the ecological distinct groupings in this study, the failed infected implant had lowest alpha diversity (Table 3) (Shannon Wiener exponential H) and the most genera distinct from the health control.Dental implant failure as a dual biofilm infection of implant surface accompanied by chronic osteomyelitic biofilm infection of bone (Zimmerli, 2014) achieved the most significant ecological pathogenicity, with 39 genera distinct from the 51 genera of the health control.Osteomyelitis is defined when an infective microbe establishes chronicity.This is characterised by low grade sessile bacterial persistence and bony necrosis (Lew and Waldvogel, 2004).
Failure to eliminate even the smallest areas of necrotic bone areas may maintain infection for years (Ochsner, 2006).The presence of a residual and persistent sclerotically confined biofilm nidus in apparently healed bone is often in subclinical form that is highly recalcitrant and may persist for life (Percival et al., 2011).Cierny (2011) describes a transition from a "dirty wound" to a "clean wound".Weinstein and Darouiche (2001) describes a similar transition of the wound as progression from pathogenic to less pathogenic and nonpathogenic.Shade et al. (2012) describes each of the states including those with high relative abundance of known pathogens as "alternative stable ecological states", but those closer to health are closer to their predisturbance homeostasis.It is suggested that these states are more diverse, less refractory with increased resistance and resilience to further disturbance (Zimmerli et al., 2004).
This study presents strong evidence that the human jawbone biological space is never microbially inert and benefits from the presence of a resident microbiota whose ecological resistance and resilience defends the space from disturbance, that is, pathological colonisation and chronic infection (Costerton et al., 2003).Beneficial bacteria are in far greater numbers compared to pathogens (Visick et al., 2021) and may play a role in resilient recovery of cellular homeostasis by using sRNA to dampen the immune inflammatory state (Moriano-Gutierrez et al., 2020).Stable biofilm communities may play the dominant role in developing normal habitat physiology and they will provide the dominant numbers of beneficial planktonic bacterial cells that engage in the "race for the surface" (Gristina et al., 1988) with pluripotent stem cells for the implant surface.Those bacteria found to be "normal microbiota" contributing to host tissue health in a resident symbiotic homeostasis may create a lasting, persistent, beneficial association (Visick et al., 2021), essential for an enduring osseointegration.Ecological diversity provides stability in the face of disturbance events and spatiotemporal change within the biofilm, minimizing the probability of extinctions (Botton et al., 2006, Kolenbrander, 2011).

Limitations and Future Directions
The authors acknowledge that further research is required to corroborate our findings and to further examine both CAB and other bone qualities for their resident microbial ecology.One method of RSD is described in this study; however, other methods may be as or more effective in recovering jawbone to microbial ecologic health.

Fig. 1 :Fig. 3 :
Fig. 1: Example of an initial non-constrained Non-ParametricMultidimensional scaling analysis in 2D (NMDS), with the bray-curtis similarity measure, revealed the possibility of six ecologically distinct groups in the sample data set.A clear cluster of significantly different groups was evident (3D stress = 0.17)

Fig. 5 :Fig. 6 :
Fig. 5: Results of an NMDS ordination.Congenitally absent tooth sites from two patients separated into two microbially distinct groups, Control CAB1 and Control CAB2

Fig. 7 :
Fig. 7: Longitudinal pie charts constructed from pyrosequencing of constituent genera in a same-site implant osteotomy: 54F-initial osteotomy in poor quality, osteoporotic, soft, non-debrided D4 bone at first implant installation.Note relatively low diversity.56E-shortterm infective implant failure.Note very low diversity, close to single genus biofilm pathology.91E-following RSD there is a return to ecologic diversity and evenness, confirmed by univariate exponential H statistical analysis.This results in improved bone quality with a return to normal histologic bony trabecular and cortical architecture (H3).Successful same-site revision followed the eradication of biofilm disease

Fig. 13 :Fig. 14 :
Fig. 13: SEM image of a bone sample taken at 9 months post-extraction, with pathological, sessile, invasive rod-bacteriaspreading over the bone surface with patchy areas of biofilm (7500).Following RSD at 9 months, at 12 months a population-shift to coccoid Bacteria (Ba) is noted, indicating that the bone-bed supports living microbial Biofilm (BF) populations(Nelson et al., 2023)

Table 3 :
Alpha diversity (exponential H) of CAB versus other samples; CAB health-control as an ecological diversity/stability reference exponential H.Note CAB has the highest alpha diversity, nearly 4 times that of a failed implant No. of patients No. of samples No. of phyla No. of genus No. of species Alpha diversity (Exp.H)

Table 4 :
(Viljoen, 2019)onential H Alpha diversity of samples 54F, 56E and 91E; longitudinal same site univariate exponential H alpha diversity and microbiological analysis of the above three same site samples, showing alpha H diversity, phyla, genera and a debrided population-shift and bone quality recovery from D4 bone to H3(Viljoen, 2019)bone with significant increase in ecologic diversity in health