This study, for the first time, provided the stringently determined network of genes that were associated with success and failure of implant osseointegration. The novel design of the present investigation was to comparatively evaluate the whole genome microarray associated with implant placement in vitamin D-deficient and control rats. Initially, we anticipated finding genes that were involved in the classic category of bone remodeling; however, in two-way ANOVA, we found NPAS2 as the most significantly affected gene by vitamin D deficiency in the peri-implant tissue. NPAS2 is a member of the basic helix-loop-helix-PAS family of transcription factors, involved in the regulation of circadian rhythm 
. The primary molecular target of NPAS2 is Per2, which was also found to be significantly modulated by vitamin D deficiency in the peri-implant tissue ().
Furthermore, KEGG () pathway analyses also highlighted the potential involvement of the circadian rhythm system in the establishment of osseointegration. Circadian oscillations are generated by a core set of genes: NPAS2, Per2 as well as ARNTL/Bmal1 that are part of a negative feedback loop and are referred to as “clock” genes 
. These genes are widely expressed throughout the body tissues and are likely involved the temporal patterning of local gene transcription 
. In the present study, NPAS2 was upregulated in the peri-implant tissue, which was completely attenuated under the vitamin D-deficient condition. In contrast, the vitamin D-deficient environment significantly increased the expression of Per2 (). The reevaluation of microarray data of ITV+ and ITV- groups by t
-test further identified Arntl/Bmal1 that was significantly downregulated in the ITV- group ().
Previous work has shown the rhythmic expression of clock genes in osteoblasts and found that mutant mice lacking these clock genes exhibit altered osteoblast proliferation 
. Other studies have found evidence for circadian regulation of cell cycle and cell proliferation 
. In the present study, we found the evidence that the placement of the implant into bone alters the expression of genes involved in circadian regulation. The experimental implant was fabricated from Ti alloy with submicron surface topography. Approximately 50% of the implant surface was further modified by HA nanoparticles 
. Ti implants have been long considered bio-inert; however, recently, Ogawa and colleagues have demonstrated that UV treatment of Ti implant can activate the surface energy, which underscores the functional involvement of electrostatic mechanism 
. The implant surface further contains complex carbon molecules such as polyaromatic hydrocarbons (PAHs) 
. It may be possible that the implant material could induce biological responses such as electrostatic or xenobiotics responses. NPAS2, Per2 and Arntl/Bmal1 responsible for circadian rhythm control contain PAS (Per-Arnt-Sim) motifs. It has been suggested that PAS-containing transcription factors are also responsible for the adaptation to environmental cues such as the metabolism of xenobiotics and hypoxia signaling 
. It is tempting to speculate that the microenvironment induced by the placement of implant may influence the disproportionate up-regulation of circadian rhythm genes, in particular NPAS2.
More surprising observation was the extent to which vitamin D deficiency also profoundly altered the expression of circadian rhythm-related genes. Previous work has found evidence that transcript for a vitamin D receptor is expressed with a daily rhythm, at least in brown adipose tissue 
. Still, the pervasive impact of implant placement and vitamin D deficiency on circadian rhythm-related gene expression in the bone matrix is a novel finding.
Hierarchical clusterization is a holistic approach based on the calculation and comparison of pairwise correlation coefficients between two datasets to arrange genes according to similarity or closeness in expression pattern 
. NPAS2 and seemingly unrelated cartilage ECM genes such as col2a1, aggrecan, and col10a1 were co-localized in a small cluster (). This finding was unexpected, but could suggest a novel co-regulatory mechanism affecting skeletal circadian rhythm system and cartilage ECM gene expression. Bone wound healing associated with implant osseointegration undergoes intramembranous bone formation without cartilage precursor tissue 
; therefore the expression of cartilage ECM molecules during osseointegration was a puzzling observation. RNA samples used for microarray assay were reevaluated by RTPCR and confirmed the expression of cartilage matrix ECM genes (); however, histological cartilage tissue was not formed in the inner chamber of T-shaped implant ().
Under hypoxic in vitro
conditions, the expression of cartilage ECM genes was reported to increase in bone marrow mesenchymal stem cells, which expressed Sox9, an initiation factor of chondrogenic differentiation 
. Hypoxia-induced factor 1 alpha (HIF-1a) has been shown to mediate the hypoxia-induced chondrogenic differentiation 
. Thus, we initially postulated that implant placement might induce a relatively hypoxic local microenvironment, inducing the observed upregulation of cartilage ECM genes. However, reevaluation of the microarray data revealed that the expression levels of HIF-1a and Sox9 were not significantly affected in all groups (). Thus, the upregulation of cartilage ECM genes associated with implant healing may employ different mechanisms.
The expression of cartilage ECM during the establishment of osseointegration has not been reported. We designed and performed a proof-of-concept in vitro
experiment to determine if the placement of the implant affects the expression of cartilage ECM. Mouse bone marrow D1 cells, when cultured on implant disk with 1,25D supplementation, significantly accelerated the expression of type II and type X collagens (). Although the expression pattern of NPAS2 in this in vitro
system did not match with those of type II and type X collagens (), the siRNA-derived knockdown study indicated that NPAS2 appeared to regulate their expression (). Circadian rhythm molecule ARNTL/Bmal1 is the obligatory partner of NPAS2 to induce target genes through a cis-acting MOP3/MOP4 responsive element (M34RE; CACGTGACC) 
. It has been reported that type II and type X collagen genes as well as aggrecan gene contain active M34RE 
. As such, we postulate that the ectopic synthesis of cartilage ECM molecules around the implant may be regulated by the activated NPAS2-ARNTL/Bmal1 heterodimer. However, an unexpected discrepancy was found in the type X collagen expression modulated by NPAS2 knockdown when D1 cells were cultured without implant. We speculate that NPAS2-ARNTL/BmalI may regulate this gene as either an enhancer or a silencer and the implant placement may disproportionately activate its anabolic function.
The presence of type X collagen protein associated with implant substrate in vitro
() and in vivo
() further provides the supporting evidence that the presence of implant material may alter the cellular behavior and induce the ectopic synthesis of cartilage ECM molecules. With 1,25D supplementation, D1 cells on implant disk further spread to form more polygonal than spindle shapes. Moreover, the type X collagen found in the presumptive bone-implant interface showed a well-defined hexagonal structure (). Kwan et al. (1991) reported that type X collagen assembled into a mat-like structure in vitro
, and the prolonged incubation resulted in the formation of a hexagonal lattice 
. Therefore, although macromolecular organization of type X collagen has not been reported in vivo
, the ECM structure found in the presumptive bone-implant interface may indeed contain type X collagen. We speculate that the postulated ectopic synthesis of cartilage ECM may play a previously unreported role in the establishment of osseointegration.
The present genome-wide microarray study of implant ossoeintegation suggests that the specific microenvironment induced by implant placement significantly affect a spectrum of gene expression networks, possibly including peripheral circadian rhythm mechanisms. The potential interaction between NPAS2 and cartilage matrix genes has led to a hypothetical mechanistic model of osseointegration that through the circadian rhythm-related system, bone marrow mesenchymal cells may initiate ectopic synthesis of cartilage matrix molecules including type X collagen without the formation of in situ cartilage tissue. Vitamin D deficiency negatively affects these processes, resulting in the lack of bone and implant integration. Taken together, we postulate that the establishment of osseointegration requires the peripheral circadian rhythm system, which transiently activates the synthesis of a selected set of cartilage matrix molecules possibly guiding the intimate integration of implant surface and bone tissue.