Octacalcium phosphate

Calcium phosphate was discovered by Johan Gottlieb in 1769, and since its discovery calcium phosphate has been widely researched and has been found to be one of the most important inorganic structures within hard tissue of vertebrates.^[3] Calcium phosphate has been used to treat various illnesses such as rickets, scrofula, diarrhea, ulcerations, and inflammation, but its applications in orthopedics and dentistry has been the main area of focus for many years.^[3] Prior to the utilization of calcium phosphates in orthopedics, bioceramics were widely utilized due to their bio inertness and advantageous mechanical properties, but despite the success of bioceramics, this material simply substituted broken bones, and did not provide a means of bone regrowth within the damaged tissue.^[3][4]

By the 1900s scientists had started utilizing calcium phosphate during surgeries as a means of applying simple bone grafts, and by 1950 the genesis of self setting calcium phosphate in combination with bioceramics had been discovered.^[3][4] After that, between 1976 and 1981 calcium phosphates had started to be utilized more prominently as coatings for orthopedic and dental implants in order to stimulate stronger osseointegration, and by the 1990s calcium phosphate had started to become utilized as an effective mode for drug transportation and had started to branch into other fields such as tissue engineering.

Octacalcium phosphate (OCP) was first discovered in the 1950s when scientists discovered that by varying the calcium phosphate ratio various forms of calcium phosphates could be created.^[4] OCP has widely been seen as an inorganic precursor for hydroxyapatite which is similar to calcium phosphate in that it is an inorganic mineral found in bones and teeth that plays a major role in the overall structure, strength, and regeneration capabilities of bone.^[5][6] Along with this, compared to other forms of calcium phosphate OCP has been found to have greater levels of biocompatibility and increased rates of osteointegration.^[5] The advantageous properties of OCP have made it a primary candidate for many orthopedic uses, and although mass production has been utilized, extremely strict chemical constraints make it difficult to mass-produce and fast paces.^[7]

Ceramics can be categorized into four categories based on their interactions with tissues. Type #1 (dense, nonporous, and inert) ceramics are strong, stiff, and attach to bone/tissue resulting in a cementing of the device into the tissue. Type #2 (porous and inert) ceramics exhibit a lower overall strength but are useful as coatings and result in biological fixation. Type #3 (dense and nonporous) ceramics exhibit biological fixation by chemically attaching directly to bone. Finally, type #4 (dense, nonporous, and resorbable) ceramics are slowly replaced with bone. The nature of octacalcium phosphate resembles that of type #4 ceramics.

Type #4 ceramics differ based on the ratio of calcium to phosphate (Ca:P), with the most stable/ideal ratio (Ca:P=10:6=1.67) resulting in hydroxyapatite (HA) which is often used in many orthopedic settings due to the inherent biocompatibility and similarity to natural bone tissue.^[8] While HA has been widely used and established as an excellent candidate for orthopedic usage, OCP (Ca:P=1.33), while harder to synthesize and more difficult to sinter and mold, has been proven to not only be more resorbable than HA, but also proven to result in greater overall bone formation than HA.^[9][10][11]

The table below displays various octacalcium phosphate material properties and descriptions of said properties.

The three crystal types (spherule, ribbon like, and plate) all exhibit flexural behavior with some displaying brittle characteristics and others displaying ductile characteristics. Spherule and Ribbon like crystals display brittle characteristics, similar to ceramics, deforming elastically up to a maximum stress and then immediately fracturing (irreversible deformation).^[13] Plate crystals however displayed more ductile characteristics. Unlike spherule and ribbon like crystals, plate crystals deformed elastically up to the maximum stress, but did not fracture, instead transitioning into plastic deformation similar to metals and some polymers.^[13]

Due to the multitude of implications of octacalcium phosphate (OCP), many synthesis methods have been developed as well as strides to upscale the overall production rate of octacalcium phosphate. Methods include precipitation reactions, hydrolysis reactions, aging, and ion substitution.^[7] Previously stated methods have all been able to produce high-purity octacalcium phosphate, but in order to upscale the production of OCP, it is imperative to control the reaction conditions as slight deviations in molarity, pH, or temperature can easily lead to different calcium phosphate variations such as dicalcium phosphate or hydroxyapatite.^[7]

Precipitation involves mixing Ca(CH₃COO)₂ (calcium acetate) with a sodium phosphate solution usually consisting of a mixture of Na₂HPO₄ (disodium phosphate) and NaH₂PO₄ (sodium hydrogen phosphate).^[15][16] The reaction constraints for precipitation reactions consisted of a calcium acetate molarity of 0.04 M, and sodium phosphate solution molarity of 0.04 M. Finally, pH levels ranged from 5.0 to 6.5 and temperature levels ranged from 37 °C - 90 °C.^[15][16]

Synthesis of octacalcium phosphate is typically done via the hydrolysis of α-tricalcium phosphate (ɑ-TCP).^[17] In order to create OCP, ɑ-TCP along with calcium carbonate and brushite (CaHPO₄·2H₂O) are formed into a solid state in preparation for the hydrolysis. The hydrolysis reaction can then be performed by combining the previously prepared ɑ-TCP and 0.0016 M phosphoric acid at 25 °C and a pH of 6. During hydrolysis reactions, in order to prevent deviation from octacalcium phosphate, it is imperative to maintain a calcium phosphate (Ca/P) ratio of 1.33.^[17][18]

Aging reactions are conducted similar to the precipitation reactions, but precipitation reactions can occasionally produce poorly defined particles due to the fast precipitation process.^[18] So, upon finishing the precipitation reaction the solution is mixed gently for times varying from 3 to 12 hours which results in well defined octacalcium phosphate crystals which can then be extracted via filtration using membrane fillers.

Ion substitution reactions are conducted similar to precipitation reactions, but instead of calcium acetate, other variations are utilized in order to result in more crystallized precipitates.^[19] Ions can include magnesium (Mg²⁺), strontium (Sr²⁺), or manganese (Mn²⁺). Varying the form of acetate that is utilized during the precipitate reactions can have varying effects depending on the element used and the concentration of element.^[19] Specifically strontium has been found to improve the bioactive properties of OCP.^[5] In terms of thermal stability the addition of strontium or magnesium into the structure can result in reduced thermal stability and increases in the extent of collapsed OCP.^[12]

Gel sponges are typically used as bone integration scaffolds mainly due to their inherent porosity. The porous structure of the gel itself can aid in osteointegration when combined with CaP ceramic composites.^[13] Gel-OCP composites can be formed using various methods, but a common method is via coprecipitation and has been known to produce optimal Gel-OCP composites while still maintaining the inherent porosity that is useful for osteointegration.^[13] In-vivo pre-clinical Studies comparing Gel-OCP composites to pure gel control groups have found that the gel scaffold is capable of regenerating substantial amounts of bone within months (~4 months) of implantation, indicating that the gel-OCP composites exhibit high osteoconductivity allowing for enhanced bone regeneration.^[13]

Collagen-OCP composites utilize collagen which is a matrix protein that accounts for 30% of total proteins within most organisms.^[20] Collagen is unique in that it can be used in many applications, such as sponges or hydrogels, or even combined with other forms of calcium phosphate such as hydroxyapatite.^[13] Along with this, collagen based composites exhibit similar properties and structure to natural bone tissue such as high osteoconductivity, and enhanced biointegration.^[13] Collagen-OCP composites, similar to gel-OCP composites, can be synthesized using numerous methods, but one common method is via molding mixtures of OCP and collagen solutions that have been extracted from animal skins.^[13] In-vivo preclinical Studies evaluating the effects of collagen-OCP composites have shown that the composite by itself displays enhanced bone regeneration, osteoconductivity, and biodegradability compared to pure OCP or collagen control group as well as stimulated osteoblast and osteoclast activity during bone regrowth and remodeling indicating potential to be used for bone regrowth in clinical applications.^[13][21][11]

Alginate is a polysaccharide derived from a form of brown seaweed that has spiked interest due to its favorable biocompatibility and its ease of gelation.^[13][22] Similar to the collagen and gel based OCP composites, both coprecipitation and mixing methods have been utilized to create alginate-OCP composites, both methods produce viable composites with favorable porosity which can be further controlled by altering the alginate concentration or centrifugal speed during synthesis reactions,^[13] Alginate-OCP composites, similar to previously stated scaffolds, have also shown increased levels of osteointegreation and osteogenic interactions as well as the ability to stimulate osteoblasts in vitro, and the ability to aid in the conversion of OCP → HA in vivo.^[13]

Hyaluronic acid is a naturally occurring polymer that is present in skin, tendons, and synovial fluid as a component of the connective tissue's extracellular matrix.^[13] As a component of composites, hyaluronic acid acts as a delivery medium for OCP.^[13] Synthesis of Hyaluronic-OCP scaffolds is achieved by simply mixing OCP granules with hyaluronic acid at a controlled pH level and results in an injectable paste.^[13] In terms of bone regeneration hyaluronic acid-OCP composite pastes have shown enhanced osteoconductivity soon after injection, and exhibited biodegradation by osteoclasts.^[13]

The structure of OCP is closely associated with HA structure, and has thus made it an attractive bone substitute for biomaterial scientists and orthopedic surgeons.^[12] A higher osteoconductivity was first observed in the bone tissue response in mouse where OCP was placed onto the calvaria in its granule form, showing it to have higher osteoconductivity than other Ca-P materials like anhydrous dicalcium phosphate (DCP), amorphous calcium phosphate (ACP), calcium deficient HA (CDHA), and stoichiometric HA.^[12] OCP also tends to biodegrade in the bone.^[12] OCP is an osteoconductive and biodegradable material capable of stimulating bone formation through osteoblast differentiation and osteoclast formation.^[12] During thermodynamic conversion of OCP to HA it was found to strongly stimulate cell capacity via hydrolysis in in-vivo environments.

Biodegradable calcium phosphates (Ca-P's) like OCP can promote bone regeneration through bone remodeling, which involves both bone resorption by osteoclasts and bone formation by osteoblasts.^[12] One study showed that osteoclast formation of OCP was almost the same as that of 𝛽-tricalcium phosphate (𝛽-TCP) and that OCP and OCP/HA mixtures had higher expression of calcium coupling factor compared to 𝛽-TCP when cultured with mouse marrow macrophages.^[12]

Activation of the bone cellular responses and stimulation of bone remodeling processes, has been shown in studies where OCP granules were implanted in mouse calvarial defects.^[12] Composite scaffolds with OCP and gelatin have also been shown to induce bone regeneration in line bones in rabbits and at faster rates than 𝛽-TCP alternatives.^[12]

Though OCP has not been established in the dental field, bioactive properties of OCP have attracted the attention of oral surgeons and researchers.^[12] For example, OCP coatings on zirconia oral implants have the potential to improve osseointegration of already existing ceramic implants due to their high osteoconductive attributes and drug delivery capabilities.^[23] This coating allowed for reproductibility, quick synthesis, simplicity, and good tensile adhesion strength. Under certain conditions, synthesis of OCP coatings may allow for incorporation of biologically active molecules in the coating, providing potential for drug delivery applications.^[23] Studies have also indicated potential for OCP-based cement as a potentially promising pulp-capping agent demonstrated in rats, concluding that OCP-based cement allowed for the occurrence of favorable healing processes in the dental pulp.^[24]

The functionalization of therapeutic agents for drug-delivery systems for the treatment of bone pathologies has focused mainly on Ca-P nanoparticles, HA nanocrystals, and apathetic cements, coatings and porous scaffolds, but literature on the use of OCP in these applications is limited.^[13] Most of this research includes functionalization of OCP with bisphosphonates (BPs), which are commonly used as antiresorptive agents.^[13] Alendronate, a commonly used BP, has been combined with OCP in some studies, demonstrating inhibited osteoclastogenesis and osteoclast differentiations but enhanced osteoblast proliferation and activity.^[13] Alendronate-loaded OCP also showed enhancement of osteoblast differentiation markers compared to HA-loaded alendronate.^[13] In-vitro tests carried out on osteoblast, osteoclast, and endothelial cell biomimetic environments showed that BPs imbue functionalized OCP with antresorptive and antitumor properties.^[13]