Peri-implant healing by de novo bone formation (increase in bone volume) involves two principal pathways, namely contact osteogenesis and distance osteogenesis as originally reported by Osborn 1979 . In contact osteogenesis (osteoconduction), predominant in trabecular bone, bone forms first on the implant surface by recruitment and migration of osteogenic cells so the implant surface, followed by bone formation in apposition to the implant surface . In distance osteogenesis, predominant in cortical bone, autochtonous new bone is formed on the surface of old bone, instead of on the implant, and approximates the implant surface from the periphery . An important factor influencing contact osteogenesis appears to be the hydrophilicity and microtopography of an implant surface (for reviews see Ref. , ). The histology of the first stages of peri-implant healing on a titanium plasma sprayed surface (TPS) was reported in 1984 by Donath et al. . However, how de novo bone formation occurs on an inert metallic surface such as titanium is unclear. Here a model is proposed for nanostructured, hyperhydrophilic titanium surfaces (see ).
2 Material and methods
Methods for the preparation of hyperhydrophilic titanium plasma sprayed surfaces are described in Ref. . The methods involved in gap healing experiments in vivo have been described for osteoinductive TPS-surfaces (BMP-2) in sheep  and for nanostructured hyperhydrophilic TPS surfaces preliminarily in minipigs . The hyperhydrophilic nanostructured TPS-implant surfaces were conserved in the dry state by an exsiccation layer of salt . They were implanted in this dry state being wetted intra operationem with blood. The de novo bone formation is defined as the ratio between the area (mm2) of new bone and the total defined area (reference region of interest) in percent. Bone ongrowth i.e. bone implant contact (BIC) via histological slide (1D) is defined as the ratio between the length (mm) of the bone-implant contact and the total defined unit length (reference, region of interest) in percent.
As has been shown by others  and in Ref. ,  ultra- and hyperhydrophilic titanium surfaces can lead to an increase in bone volume (osteoinduction) and bone ongrowth i.e. BIC (osteoconduction). This is illustrated in Table 1.
Bone volume increase and bone ongrowth increase on nanostructured surface in minipig femur (see ).
|TPS-control super-hydrophobic||TPS-CSA hyper-hydrophilic||p-Value|
|Osteoid volume (%)||1.79 ± 0.33||3.78 ± 0.34||<0.001|
|Bone ongrowth (%)||0.073 ± 1.41||6.68 ± 1.48||<0.030|
|Osteoid ongrowth (%)||0.40 ± 1.10||7.53 ± 1.01||<0.004|
a100% = 6.5 mm,
Titanium TPS surfaces are originally super- or hyperhydrophobic. A wettability shift by acid etching makes the surfaces hyperhydrophilic. In experiments on nanostructured hyperhydrophilic TPS surfaces (Table 1) 4 weeks after implantation in minipigs  osteoid volume increases two-fold corresponding to induced de novo bone. Bone ongrowth (i.e. BIC) after 8 weeks increases nearly by two orders of magnitude and osteoid ongrowth (BIC) after 12 weeks nearly 20-fold.
The question is, how can these increases in peri-implant bone volume and bone implant contact be explained?
4.1 Macrophages are the first cells to colonize a titanium implant
In 1984 Donath et al.  first described mononuclear histiocytes, today known as stationary form of connective tissue macrophages , on the surface of TPS coated titanium cylinders 3 days after implantation in the rat femur (Figure 1). Between 5 and 56 postoperative days foreign body giant cells (multinucleate macrophages) on the implant surface were observed. Two populations could be distinguished cytoplasm-rich “acid-phosphatase-positive giant cells” and smaller “acid-phophatase-negative giant cells” . In contrast osteoclasts and osteoblastswere observed on bone spicules and trabeculae  but not on the Ti-implant. The above results are in agreement with the recent conclusion that specific osteal macrophages (“osteomacs”) exist in bone and that osteal macrophages and osteoclast precursor cells are cytologically different after having diverged from a common progenitor cell . Thus the observations of Donath et al. strongly indicate the importance of macrophages for peri-implant endosseous healing.
Peri-implant healing by de novo bone formation involves two principal pathways, namely contact osteogenesis and distance osteogenesis as originally reported by Osborn 1979 . Of prime importance for bone bonding to the biomaterial surface is a structure called “cement line” an initial secretion of non-collagenous proteins followed by mineral nucleation and crystal growth , .
Of key importance appear to be the physico-chemical properties of the hyperhydrophilic CSA-TPS implant surface as previously described , . Together with the high wettability expressed in imaginary contact angles of Θ
4.2 M2-macrophages synthesize BMP-2
An important discovery was the polarization of macrophages into two phenotypes i.e. destructive inflammatory M1 and reparative wound healing M2 macrophages . Crucial growth factors of such reparative macrophages for bone growth are VEGF  and surprisingly also of BMP-2 , . Therefore the findings of oseoconductivity can be interpreted on this pathophysiological and biochemical basis. BMP-2 in concentrations of 50–300 pg/ml can be secreted into the cell culture medium by macrophage cell lines such as J774A.1 and RAW264.1 cells , . In sum these findings can be interpreted as a new pathophysiological and biochemical basis of osteoconductivity.
4.3 Nanostructures on implant surface stimulate synthesis of BMP-2
Recently Sun et al.  reported that TiO2 nanotube layers stimulate RAW 264.7 macrophages to secrete BMP-2 in contrast to smooth surfaces. A nanostructure with tubes of 30 nm in diameter begins to stimulate BMP-2 secretion versus a smooth surface. BMP-2 secretion by RAW 264.7 macrophages then increases two-fold up to 300 pg/ml as the diameter is increased to 120 nm. Since the hyperhydropilic TPS surfaces (e.g. in Table 1) are nanostructured , it is conceivable that the osteal macrophages are optimally stimulated by the nanostructured TPS surface to secrete BMP-2, explaining the osteoinductive and osteoconductive properties of this surface (Table 1). In contrast the super hydrophobic TPS controls, which lack high wettability, high wetting and spreading rates as well as the macrophage stimulating nanostructure, show practically neither osteoinductivity nor osteoconductivity. Given these results, hyperhydrophilic nanostructured implants may display a great potential in a variety of orthopedic and trauma surgery applications.
4.4 Macrophage model of osseointegration
A novel model of surface contact osteogenesis can be proposed on the above observations. The new insights indicate that the osteoconductive efficiency of hyperhydrophilic micro- and nanostructured surfaces can be tested in vitro . The putative mononuclear osteal macrophages arriving on day 3 on the implant surface as histiocytes  (Figure 2A) differentiate further into M2 wound healing macrophages putatively secreting BMP-2 (Figure 2B) for recruiting osteoblasts to the implant surface by chemotaxis. Donath et al.  in addition clearly describe multinuclear giant cells on day 5 (Figure 1). Thus the “cytoplasm-rich acid-phosphatase-positive and smaller acid-phophatase-negative giant cells , are in agreement with osteal macrophages and osteoclast precursor cells having diverged from a common progenitor cell . Recently it was shown that interleukin 4 converts mononuclear macrophages into multinuclear giant cells  (Figure 2C). At present it is unclear, how BMP-2 in concentrations of 50–300 pg/ml stimulate bone growth, when the affinity of BMP-2 receptors on bone precursor cells and osteoblasts is two to three orders of magnitude lower , . However, this might be explained by a juxtacrine secretion model  or other unconventional secretory processes .
In the model (Figure 2) it is proposed that MGCs not only secrete BMP-2 for recruiting osteoblasts to the implant surface by chemotaxis (Figure 2), but also VEGF for angiogenesis initiating osteoinduction. BMP-2 secretion is stimulated further by nanostructures offering an innovative approach to synthesizing bioactive implant surfaces. A stimulation of VEGF secretion by nanostructures has however, not yet been shown.
Research funding: The author state no funding involved. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.
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