Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated mechanisms. They have superior mechanical properties due to their complex architecture. Control over biomineral properties can be accomplished by regulation of particle size, shape, crystal orientation, and polymorphic structure. In many organisms, biogenic minerals are assembled using a transient amorphous mineral phase. Here we report that organic constituents of bones and teeth, namely type I collagen and dentin matrix protein 1 (DMP1), are effective crystal modulators. They control nucleation of calcium phosphate polymorphs and the assembly of hierarchically ordered crystalline composite material. Both full-length recombinant DMP1 and post-translationally modified native DMP1 were able to nucleate hydroxyapatite (HAP) in the presence of type I collagen. However, the N-terminal domain of DMP1 (amino acid residues 1-334) inhibited HAP formation and stabilized the amorphous phase that was formed. During the nucleation and growth process, the initially formed metastable amorphous calcium phosphate phase transformed into thermodynamically stable crystalline hydroxyapatite in a precisely controlled manner. The organic matrix-mediated controlled transformation of amorphous calcium phosphate into crystalline HAP was confirmed by x-ray diffraction, selected area electron diffraction pattern, Raman spectroscopy, and elemental analysis. The mechanical properties of the protein-mediated HAP crystals were also determined as they reflect the material structure. Such understanding of biomolecule controls on biomineralization promises new insights into the controlled synthesis of crystalline structures.

The synthesis of calcite fibers has been confined to nature and is observed most prominently in sea urchin teeth and bacterial deposits. By generating a liquid-phase mineral precursor, induced by the addition of acidic macromolecules to a crystallizing solution, calcite fibers with diameters ranging from 100 to 800 nm have been deposited on existing calcite substrate crystals. A solution-precursor-solid (SPS) mechanism, which has features similar to both the vapor-liquid-solid (VLS) and solution-liquid-solid (SLS) mechanisms, is proposed as the growth mechanism. Because this aqueous-based SPS mechanism occurs under physiological conditions (and down to temperatures as low as 4 °C), it is feasible that it may be used by organisms to form their fibrous biomineral structures. This discovery suggests an interesting link between two seemingly unrelated processes, high-temperature semiconductor fiber formation and biological mineralization.

Bone and dentin biomineralization are well regulated processes mediated by extracellular matrix proteins. It is widely believed that specific matrix proteins in these tissues modulate nucleation of apatite nanoparticles and their growth into micrometer sized crystals via molecular recognition at the protein-mineral interface. However, this assumption has been supported only circumstantially and the exact mechanism remains unknown. Dentin matrix protein 1 (DMP1) is an acidic matrix protein, present in the mineralized matrix of bone and dentin. In the present study we have demonstrated using synchrotron small-angle X-ray scattering that DMP1 in solution can undergo oligomerization and temporarily stabilize the newly formed calcium phosphate nanoparticle precursors by sequestering them and preventing their further aggregation and precipitation. The solution structure represents the first low resolution structural information for DMP1. Atomic force microscopy and transmission electron microscopy studies further confirmed that the nascent calcium phosphate nuclei formed in solution were assembled into ordered protein-mineral complexes with the aid of oligomerized DMP1, recombinant and native. This study reveals a novel mechanism by which DMP1 might facilitate initiation of mineral nucleation at specific sites during bone and dentin mineralization and prevent spontaneous calcium phosphate precipitation in areas in which mineralization is not desirable. KeywordsDentin Matrix Protein 1; Biomineralization; SAXS Mineralized tissue such as bone and dentin are unique biocomposites of a structured organic matrix impregnated with matrix-oriented carbonated apatite crystals (1). The rigidity and compressive strength of bone and dentin are directly dependent upon temporally and spatially controlled mineral nucleation and hierarchically assembled matrix (2,3). It is well known that biological fluids are metastable or supersaturated with respect to calcium salts i.e., they are below the saturation needed for spontaneous precipitation but are well above the saturation to support crystal growth after the initial nucleus has formed (4,5). Therefore, a driving force is required to lower the activation energy of nucleation and formation of thermodynamically To whom correspondence should be addressed. (A.G) Compelling in-vitro and in-vivo data provide evidence that bone and dentin specific noncollagenous proteins play a critical role in the initiation and growth of the calcium phosphate mineral phase (6-8). Although the exact mechanism remains unknown, it is postulated that kinetic control of biomineral crystallization is achieved by interactions between mineral nuclei and soluble or immobilized acidic proteins. Atomic force microscopy indicates that proteins extracted from calcite or aragonite-containing layers of the abalone shell bind to growth sides on well-defined calcium carbonate crystal surfaces and influence the kinetics of crystal growth from solution (9). This protein-mineral interaction might be an essential strate...

Dentin mineralization requires transcriptional mechanisms to induce a cascade of gene expression for progressive development of the odontoblast phenotype. During cytodifferentiation of odontoblasts there is a constant change of actively transcribed genes. Thus, tissue-specific matrix genes that are silenced in early differentiation are expressed during the terminal differentiation process. Dentin sialophosphoprotein (DSPP) is an extracellular matrix, prototypical dentin, and a bone-specific gene, however, the molecular mechanisms by which it is temporally and spatially regulated are not clear. In this report, we demonstrate that dentin matrix protein 1 (DMP1), which is localized in the nucleus during early differentiation of odontoblasts, is able to bind specifically with the DSPP promoter and activate its transcription. We have identified the specific promoter sequence that binds specifically to the carboxyl end of DMP1. The DNA binding domain in DMP1 resides between amino acids 420 and 489. A chromatin immunoprecipitation assay confirmed the in vivo association of DMP1 with the DSPP promoter. Interactions between DMP1 and DSPP promoter thus provide the foundation to understand how DMP1 regulates the expression of the DSPP gene.Odontoblast differentiation is a well designed process that couples cell-cycle withdrawal with the synthesis of dentin matrix proteins. A number of macromolecules are involved in dentin mineralization. One such protein, dentin sialophosphoprotein (DSPP) 2 is produced by terminally differentiated odontoblasts and is known to modulate dentin mineralization. DSPP is a compound gene encoding for two proteins namely DSP (dentin sialoprotein) and PP (phosphophoryn) (1). Earlier reports have indicated DSPP as the sole odontoblast-specific gene (2); however, a recent report demonstrates low levels of expression in osteoblasts and calvarial tissue (3).Tissue-specific gene expression is complex and, among many events, requires the orderly binding of transcription factors to specific gene regulatory sequences, alteration of chromatin structure by acetyltransferases and demethylases, and recruitment of the RNA polymerase II complex (4). The temporal and spatial expression of DSPP is tightly regulated by the transcriptional elements in the promoter (5). In an attempt to identify the regulatory elements controlling the temporal and spatial expression of DSPP, a DSPP promoter of ϳ1.7 kb has been cloned and characterized to a certain extent (6). Deletion mapping experiments indicated the presence of various regulatory sequences (7). The activity of this promoter has been demonstrated by transgenic technology with a reporter construct expressing LacZ under the control of the DSPP promoter (5). It was reported that DSPP transcription is regulated by several positive and negative regulators of gene transcription in combination with the various signaling pathways/networks. Some of the negative factors known to down-regulate DSPP promoter activity have been reported earlier, including transforming growth f...

Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the selfassembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the selfassembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.

Gene therapy-based treatment such as optogenetics offers a potentially powerful way to bypass damaged photoreceptors in retinal degenerative diseases and use the remaining retinal cells for functionalization to achieve photosensitivity. However, current approaches of optogenetic treatment rely on opsins that require high intensity light for activation thus adding to the challenge for use as part of a wearable device. Here, we report AAV2 assisted delivery of highly photosensitive multicharacteristic opsin (MCO1) into ON-bipolar cells of mice with retinal degeneration to allow activation by ambient light. Rigorous characterization of delivery efficacy by different doses of AAV2 carrying MCO1 (vMCO1) into targeted cells showed durable expression over 6 months after delivery as measured by reporter expression. The enduring MCO1 expression was correlated with the significantly improved behavioral outcome, that was longitudinally measured by visual water-maze and optomotor assays. The pro/anti-inflammatory cytokine levels in plasma and vitreous humor of the vMCO1injected group did not change significantly from baseline or control group. Furthermore, biodistribution studies at various time points after injection in animal groups injected with different doses of vMCO1 showed non-detectable vector copies in non-targeted tissues. Immunohistochemistry of vMCO1 transfected retinal tissues showed bipolar specific expression of MCO1 and the absence of immune/inflammatory response. Furthermore, ocular imaging using SD-OCT showed no change in the structural architecture of vMCO1-injected eyes. Induction of ambient light responsiveness to remaining healthy bipolar cells in subjects with retinal degeneration will allow the retinal circuitry to gain visual acuity without requiring an active stimulation device.

Retinal degenerative diseases, such as retinitis pigmentosa (RP) and dry age-related macular degeneration, have led to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available, although optogenetic therapies are in clinical development. We demonstrate vision restoration using multicharacteristics opsin (MCO1) in animal models with degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of adeno-associated virus (vMCO1), leading to significant improvement in visually guided behavior conducted using a radial arm water maze. The time to reach the platform and the number of error arms decreased significantly after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for channelrhodopsin-2 opsin. Viability of vMCO1-treated retina is not compromised by chronic light exposure. Safe virus-mediated MCO1 delivery has potential for effective gene therapy of diverse retinal degenerations in patients.

Retinal degenerative diseases, such as retinitis pigmentosa (RP) and dry age-related macular degeneration, have led to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available, although optogenetic therapies are in clinical development. We demonstrate vision restoration using multicharacteristics opsin (MCO1) in animal models with degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of adeno-associated virus (vMCO1), leading to significant improvement in visually guided behavior conducted using a radial arm water maze. The time to reach the platform and the number of error arms decreased significantly after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for channelrhodopsin-2 opsin. Viability of vMCO1-treated retina is not compromised by chronic light exposure. Safe virus-mediated MCO1 delivery has potential for effective gene therapy of diverse retinal degenerations in patients.