This work determined that substantial restoration of the mechanical properties of hydrated carious dentin tissue occurred with the PILP process, thus providing functional remineralization 
in artificial lesions. Previously it has been observed that during demineralization the mineral is removed much more slowly from within the collagen fibrils than from the extrafibrillar compartment 
. Mineral remaining within the collagen fibrils could act as nuclei for regrowth of the mineral and restoration of dentin properties 
Restoration of properties was evaluated using nanoindentations performed on the hydrated tissue, since dehydration alters the mechanical response of the collagenous matrix and provides little information about the functionality of dentin 
. When carious or partially demineralized dentin is measured dry, the demineralized portion collapses and its measurement gives erroneously high values. Those values correlate linearly with total mineral content, but do not reflect the actual loss of stiffness and hardness due to missing mineral and lack of mineral coupling to the organic matrix. Thus impaired functionality of the tissue becomes evident when properties are measured under hydrated conditions, the natural state of dentin 
The changes in ER
of hydrated artificial caries specimens prepared using an acetate buffer (pH 5) that produces a sloped mineral profile similar to natural lesions were evaluated, although our artificial lesions had a larger and more pronounced outer zone of very low mechanical properties as compared to the natural caries previously studied 
. Artificial lesions offer significant advantages over natural lesions, since they are reproducible, and control lesions can be evaluated as well as the remineralized lesions. 140 μm lesions were used that provide sufficient depth to allow evaluation of remineralization changes at various time points.
When remineralized using the polymer-induced liquid-precursor (PILP) process the artificial lesions gradually recovered their mineral content and also recovered substantial mechanical properties that varied with location in the lesion, and included full recovery in the deeper portion and less recovery in the originally more demineralized portion. ( and supplementary Fig. S1
). Property gains initially were most rapid in the gradient portion of the lesion and increased towards the surface with increased mineralization time between 7–14 days with smaller changes thereafter (). When shallower artificial lesions were remineralized without the polyaspartic acid additive, only minor mechanical properties improvement were observed at the surface, and often areas of mineral precipitation were apparent 
. When 140 µm lesions were treated in the same way for 14 days only minor improvement was seen in the sloped portion of the specimens. In addition, SEM and EDS analysis showed clusters of HA crystals formed on the surface of the matrix when polyaspartic acid was not included. In contrast, no such precipitates formed on the matrix when the PILP process was used 
. This strongly suggests that new mineral was deposited within the intrafibrillar and extrafibrillar portions of the matrix.
The averaged mineral profiles from MicroXCT™ indicated restoration of mineral starting from the deepest part of the lesion and providing nearly complete remineralization after 14 days with only minor additional gains in the following 14 days (). When the lesions' nanomechanical properties were analyzed in cross-sections, a gradient was observed in ER and H, with properties decreasing from the normal tissue values along a slope towards the surface of the lesion similar to the mineral profile of the initial lesions. PILP remineralization led to recovery of the properties fairly rapidly within the first 7 days in a 70 µm zone close to the base or inner portion of the lesion, while stiffness increased in the rest of the lesion at a slower rate and reached only 50%–60% of normal tissue values with 14 or 28 days of remineralization. SEM showed distinct differences in the microstructure of the remineralized lesions () with tubules nearly filled and covered in the outer zone, and the periphery of the tubules showing a lip of mineral. Although EDS failed to show a difference in calcium or phosphate content between the two regions, both the remineralized outer and gradient zones were structurally distinct from normal dentin or the artificial caries lesion. Ultrastructural investigation by TEM () showed that the collagenous matrix at the acid-etched lesion sites was severely demineralized but contained some residual minerals; only after 14 days of PILP remineralization, collagen fibrils filled completely with organized plate-like apatite crystals and showed characteristic 67-nm type-I collagen D-bands. The crystallinity and alignment of intrafibrillar minerals increased with growth time. The collagen fibrils in outer portions of the lesions were not fully mineralized even after 14 days, although total mineral content was normal by Micro XCT, which may account for the weaker mechanical properties. This suggests that treatment length and lesion depth may affect the potential of dentin to be functionally remineralized.
Two zones existed within the artificial lesions that may interact differently with remineralization solutions. An inner zone at the bottom of the lesion is more readily remineralizable and fully recovers its properties at a rapid rate, while an outer zone requires additional time to improve in its mechanical response. Although mineral levels returned to normal in this outer zone, the properties did not fully recover at the longest time evaluated. Thus the mineral in this outer zone may not have been sufficiently incorporated into the collagen fibrils to provide complete recovery. This result might also reflect damage to the remaining collagen matrix in this outer zone that was severely demineralized. Several possibilities exist that we are currently investigating. Firstly, it has been established that dentin contains inactive matrix metaloproteinases 
. and cysteine cathespins 
that may be activated as mineral is lost due to demineralization at pH 5. Thus some of the collagen fibrils may be irreversibly damaged and therefore could not be completely functionally remineralized. In the inner zone the demineralization is much less severe and the remaining mineral prevents the protease activation. The use of protease inhibitors may alleviate this problem 
. A second possibility is that removal of nearly all mineral in this zone allows for matrix expansion as water replaces mineral and the original organization is not fully recovered during demineralization. This idea is supported by TEM images showing a less dense matrix after remineralization of the outer zone (data not shown). A further possibility is that the intrafibrillar to extrafibrillar mineral ratios or crystallite sizes are not fully recovered. Each of these possibilities needs further investigation. Thus additional studies are warranted to identify structural differences in these two zones. Each zone may require a different strategy for functional remineralization. The highly demineralized superficial zone and partially demineralized zone represented by the regions of increased slope in the artificial lesion profiles are likely to correspond to the intensely stained zone, and less stained inner zones, respectively, that are identified by caries detector in clinical caries 
There was a significant discrepancy between mineral profiles and the mechanical properties profiles at each time point. The lesions measured by mineral profile appeared shallower, particularly in the outer zone as compared to mechanical properties profiles using ER and H. This suggests that the main difference was in this outer zone and depended on the difference in detection of the two methods. It is likely that the lesions appear deeper by nanoindentation because a critical mineral level must be reached before the indentation force can be resisted. In addition it should be noted that the two methods utilized on the same specimens do not measure exactly the same locations or tissue volumes as illustrated in .
Within the limitations of the present experiments, the following could be concluded: functional remineralization of partially demineralized human dentin occurred with recovery of mechanical properties, with progressive intra- and extra-fibrillar mineralization initiated in the depth of the lesion. The degree of remineralization increased with time over the 4 week treatment period. Approximately half of the lesion depth (in a total depth of 140 µm) recovered to normal levels of ER, while the outer portion recovered about 50% of its mechanical properties during the remineralization period, even though normal mineral recovery was achieved. Results suggest that functional remineralization through shallow lesions is possible within 2–4 weeks, demonstrating the clinical translation potential of the proposed mechanism.