Current Application of β‐tricalcium Phosphate Composites in Orthopaedics

Abstract

Presently, bioceramic materials have been extensively used in spinal surgery as bone grafts; however, there are some limitations for bioceramic materials. Calcium sulfate is rapidly absorbed in vivo, the degradation of which often occurs prior to the formation of new bones. Hydroxyapatite (HA) is hardly absorbed, which blocks the formation of new bones and remodeling, and results in poor local stability or permanent stress concentration. Only β‐tricalcium phosphate (β‐TCP) is relatively balanced between scaffold absorption and bone formation. And it is a good biodegradable ceramic material that could supply a large quantity of calcium ion and sulfate ion as well as scaffold structure for bone regeneration. However, the problem of single β‐TCP is lack of osteoinductivity and osteogenicity, which restricts its application. Therefore β‐TCP composite materials have been used in the field of orthopaedics in recent decades, which fully use excellent properties of other bone repairing materials, such as biodegradability, osteoinductivity, osteogenicity and osteoconductivity. These materials make up for the deficiencies of single β‐TCP and endow β‐TCP with more biological and physical properties.

In the last 100 years, several bioceramic materials, including β‐tricalcium phosphate (β‐TCP), calcium sulfate, and hydroxyapatite (HA) have been extensively used in orthopaedic surgery as bone grafts1; however, there are some limitations for bioceramic materials. For example, the absorption of calcium sulfate is rapid in vivo, the degradation of which often occurs prior to the formation of new bones. HA is hardly absorbed within the body, blocking the formation of new bones and remodeling2, which results in poor local stability or permanent stress concentration3. β‐TCP is relatively balanced between absorption and new bone formation, and also can release a large quantity of calcium ion (Ca²⁺) and sulfate ion (SO₄ ²⁻) as indispensable inorganic salts for new bone formation4, 5, meanwhile preserving structural stability.

When used as an osteoconductive material; however, β‐TCP has quite a few disadvantages. First, its absorption does not completely agree with the new bone absorption. In general, the former is slightly faster than the latter6. Second, its mechanical properties are poor with slight brittleness, which makes it unable to resist against fatigue and insufficient holding power, thereby making it susceptible to scaffold collapse or internal fracture7, which restrains its application in weight‐bearing areas8. In addition, despite spatial scaffold providing excellent osteoinductivity, β‐TCP lacks osteoinductivity and osteogenicity9, 10. In vivo studies have suggested that the osteogenesis effect of β‐TCP is very limited7, 11. Therefore, the disadvantages of β‐TCP have restricted its application in clinical practice.

In order to overcome the disadvantages of β‐TCP, some bone‐repairing materials have been used to form β‐TCP composite materials for improving the biological and physical properties of β‐TCP. These bone‐repairing materials include bone‐induced materials (bone morphogenetic protein‐2 [BMP‐2], platelet‐rich plasma [PRP]), osteogenic materials (mesenchymal stem cell [MSC] and bone marrow), and osteoconductive materials (poly‐caprolactone [PCL] and HA). In addition, some metal ions such as Si and Zn can also be added to regulate β‐TCP degradation by activating or inhibiting the activity of osteoclasts or osteoblasts. In this study, the biological properties and research progress of β‐TCP composite materials was analyzed.

β‐TCP + Recombinant Human BMP‐2 (rhBMP‐2)

RhBMP‐2 is an osteoinductive material, which belongs to the transforming growth factor‐β (TGF‐β) superfamily. RhBMP‐2 can promote the differentiation of multipotent MSCs to osteoblasts and secretion of extracellular matrix (ECM)12. Therefore, it can induce the formation of heterotopic bones and cartilages13, and shorten the duration of bone healing. However, rhBMP‐2 has a very short half life. As a consequence, it is limited within the body. Therefore, a carrier is required to slowly release BMP, resulting in the promotion of bone growth throughout the bone's healing period.

For β‐TCP + BMP composite, β‐TCP, as a carrier, can prolong the action time of BMP, on the other hand, BMP can make up for the deficiency of osteoconductivity of β‐TCP and accelerate the formation of new bones. Urist et al.14 reported that β‐TCP possibly stimulated the biological activity of BMP, because the new bone mass under β‐TCP + BMP (1 mg) was 12 times that of BMP alone. Dohzono et al.15 found that the surface of β‐TCP scaffold contained BMP‐2 owned massive osteoclasts. In other words, BMP can accelerate the degradation of β‐TCP scaffold, which provide sufficient space for new bone formation and remodeling. Ohyama et al.16 reported that the β‐TCP + BMP composite produced better interbody fusion rate and mechanical strength, and more bone trabecula in posterior lumbar fusion of dogs, compared with β‐TCP alone, autograft, and untreated groups. In addition, β‐TCP blocks the invasion of surrounding soft tissues, thereby providing favorable conditions for the formation of new bones. Besides prolonging releasing of BMP, β‐TCP may also activate rhBMP‐217.

RhBMP‐7 is another member of the BMP subgroup, which is also termed as the bone morphogenic protein, having major effects of promoting proliferation and differentiation of osteoblasts and osteoclasts18, 19. Presently, the main carrier of rhBMP‐7 is the collagen sponge20, 21. However, in terms of mechanical properties, the collagen sponge is poor and hardly forms a stable fusion following surgery22. In contrast, β‐TCP can resist the compression from surrounding soft tissues to form good stability23. Therefore, β‐TCP possesses more excellent scaffold action than collagen, which means that β‐TCP will become more popular as a carrier in the future.

β‐TCP + HA

Both β‐TCP and HA are osteoconductive but differ in their bioactivities. The former has rapid absorption speed and high hydrophilia, whereas the latter has poor absorption, high brittleness, and poor mechanical properties24. Animal experiments have found that β‐TCP or HA alone are accompanied with several postoperative complications. Shima et al.25 used β‐TCP alone in animal models with intervertebral discectomy and fusion, and found that 70% of β‐TCP scaffolds developed fracture or displacement, with 50% developing spinal compression. Cook et al.26 adopted single HA material in canine cervical spinal fusion, finding that 39% showed graft failure at postoperative 24 weeks, leading to intervertebral height loss and limiting intervertebral fusion rate.

The β‐TCP/HA composite material is also named as biphasic calcium phosphate (BCP). This composite can increase the mechanical strength of ceramic materials and improve the degradation of β‐TCP, which ensures the stability of scaffold material prior to new bone formation27. Angela et al.1 found that BCP absorbs massive osteoblasts into the scaffold to proliferate and differentiate, and also promotes the osteoblast activity and helps the new bone formation, while single HA did not do the same. Wongwitwichot et al.24 found that BCP owned stronger surface activity, osteogenic capability, and mechanical properties. After soaking in the alkaline buffer solution for 2 weeks, BCP could form apatite‐like deposits on the surface of scaffold, which was beneficial for cell adhesion and proliferation. In histological tests, new bones were found within the BCP scaffold and surrounded with massive osteoblasts and ECM. On the other hand, fewer new bones were observed among single β‐TCP, which was primarily positioned within the outermost scaffold. In terms of flexural strength, BCP was also significantly superior to β‐TCP (25 + 1.2 MPa vs 64.5 + 18 MPa). Jeffrey et al.28 fabricated three kinds of BCP ceramics with different porosity (30%, 50% and 70%), as a result, all of them produced a significantly higher new bone mass than the autograft group. Although 50% of BCP scaffolds showed different degrees of microstructure fracture or collapse, the intervertebral height could be maintained. In the autograft group, there was not only 50% fracture but intervertebral height loss in all the animals. Therefore, they believed that BCP will be an ideal bone substitute for cervical anterior fusion.

Within BCP, contents or ratio of β‐TCP and HA could affect new bone mass and material degradation rate. Fariña et al.29 found that new bone formation was faster when β‐TCP:HA was 85:15. Bone mass analysis showed that the β‐TCP level was positively correlated with the new bone mass. In addition, the HA/β‐TCP ratio could change absorption of BCP2. Fariña et al.29 observed that BCP in the dog's body showed a faster degradation when β‐TCP:HA was 85:15, as compared with 15:85. Kwon et al.6 found that after soaking in the Ringer's solution for 1 day, HA alone did not induce any changes in the calcium and sulfate ions of the solution. In fact, β‐TCP significantly elevated the two ions over a duration of 30 days. For 1:1 BCP, the solubility was different from single HA and β‐TCP. Therefore, it can be inferred that the solubility of BCP could be regulated by the HA/β‐TCP ratio. Akamaru et al.30 believed that 60% of HA in BCP restricted the degradation speed of ceramic materials. Perhaps, it was not suitable as the carrier of BMP‐2 and the β‐TCP content of 85% was beneficial for BCP absorption.

In conclusion, β‐TCP + HA has numerous advantages. β‐TCP provides calcium and phosphorus ions, which are useful in differentiating the maturation of osteoblasts. In fact, HA provides a favorable microporous scaffold environment, which is conducive for the proliferation and differentiation of osteoblasts29. Both β‐TCP and HA could promote new bone formation. However, compared with single HA or β‐TCP, BCP could produce more new bone mass for shorter duration of time. By adjusting the β‐TCP/HA ratio, the equilibrium between ceramic material absorption and new bone formation could be maintained1, 21. This makes up for the deficiencies of too rapid absorption of single β‐TCP and hard absorption of HA. In fact, we have witnessed an improvement in the mechanical strength of ceramic materials, which was helpful in maintaining the stability of scaffolds in the fluid.

β‐TCP + MSC

Being one of three major factors in bone tissue engineering studies, MSC is derived from the bone marrow, muscles, and periosteum30, 31. In fact, it can differentiate into different types of histiocytes for regulating different growth factors32. These growth factors could promote MSC to differentiate into osteoblasts and proliferation of osteoblasts. Finally, they accomplish bone regeneration and reparation.

For the β‐TCP/MSC composite materials, MSC makes up for the deficiencies of β‐TCP in osteogenicity. Meanwhile, β‐TCP provides scaffolds necessary for differentiating and proliferating of MSC. Gupta et al.11 synthesized MSC/β‐TCP compounds with selective catalytic reduction (SCR) technique, and implanted the compound between transverse processes of goats. At postoperative 6 months, the fusion rate was 25% in both MSC/β‐TCP and autograft groups. After postoperative 12 months, the fusion rates were 33% in the β‐TCP/MSC group, 25% in autograft group, 8% in β‐TCP/BM group, and 0% in β‐TCP group. In a study conducted by Orii et al.7, β‐TCP was soaked in the MSC suspension containing medium for 1 min, and cultured for 3 h at 37°C. Electron microscopy confirmed that MSC adhered to the pore surface of β‐TCP scaffolds. The autograft bone, β‐TCP/MSC compounds, and single β‐TCP were implanted between L4‐5 transverse processes of monkeys. Their postoperative fusion rates were 67%, 83%, and 0%, respectively. Ultramicro CT showed that the new bone mass was significantly higher in the β‐TCP/MSC group than the autograft group. Bone mineral density increased in the β‐TCP/MSC group, resulting from the new bone formation, but it declined in the single β‐TCP group for the purpose of absorption. In the authors' opinion, β‐TCP/MSC induced early osteogenesis in almost all the pores of every scaffold. New bones covered the surface of scaffolds, which can effectively avoid the corrosion of scaffolds and reduce the dissolution speed of β‐TCP. Boo et al.33 described that the alkaline phosphatase and osteocalcin in the TCP/MSC group (12.3 ± 1.7 and 682.5 ± 27.7, respectively) were significantly higher than the single β‐TCP group (1.2 ± 0.7 and 5.6 ± 1.3, respectively). In addition, histological examinations suggest that new bones and matured tabular bones are detected in composite scaffolds and single fibrous tissues in single β‐TCP scaffolds. Kai et al.34 implanted MSC/BCP between L5–L6 of rabbits. As a result, compared with autograft and single ceramic material groups, the MSC/BCP group had higher stability at postoperative week 12. Jang et al.35 conducted a study to evaluate the effect of MSC on segmental bone defects of canine radius. Histomorphometric analysis revealed that the percentages of new bone formation and residual β‐TCP scaffolds were respectively 10.92 ± 2.74% and 24.21 ± 8.75% in the MSC/TCP groups, 4.08 ± 2.08% and 40.63 ± 17.86 in single β‐TCP group. Besides, MSC level could be related with new bone mass and bone healing36. Hernigou et al.37 injected bone marrow into non‐unions of the tibia, and found that the bone defect healed only in the event of the cell concentration of more than 1500/mL. However, the exact concentration of MSC continues to remain controversial.

In conclusion, β‐TCP/MSC composite has both osteogenicity and osteoconductivity, which is beneficial for new bone formation. MSCs adhere to the inner surface of β‐TCP scaffolds, thereby enabling osteogenesis on the surface and center of scaffolds. Compared with MSC, bone marrow or β‐TCP alone, the composite scaffold can induce more bone trabeculas and new bone mass, higher local fusion rate and stability.

β‐TCP + Bone Marrow

Bone marrow is one of the most important sources of MSC with osteogenicity, which was discovered for the first time by Friedenstein et al.38 in 1969. However, the fluidity of bone marrow results in difficulty to attain optimum concentration, which is necessary for new bone formation. As a consequence, single bone marrow hardly produced the osteogenic effect39. The cell/scaffold compound exhibited limited cell fluidity, thereby causing cells to adhere to ceramic scaffolds, which was proved to be quite significant for new bone formation40. Gupta et al.39 compared the effects of β‐TCP/bone marrow with single β‐TCP, bone marrow, and autoilium in lumbar posterior spinal fusion of rabbits. At the 24th postoperative week, imaging examinations revealed that the fusion rate was β‐TCP/bone marrow > β‐TCP > autograft > bone marrow. Furthermore, histological examinations suggested that lamellar bones were significantly higher in the β‐TCP/bone marrow group than the β‐TCP group. In fact, no new bones were found in the single bone marrow group. To evaluate the osteogenic properties of bone marrow, peripheral blood, and monocytes, Becker et al.41 implanted β‐TCP that was impregnated with either of the three substances in sheep models with tibial defect. Histological examinations 6 and 12 weeks postoperatively displayed most new bone mass in the β‐TCP/bone marrow group.

According to the reports provided by Cinotti et al.42, imaging examinations did not show any significant difference in the fusion rates between ceramics/MSC and bone marrow/ceramics groups. However, histological examination displayed there were thicker bone trabeculas and more bone marrow‐derived cells on the surface and center of ceramics/MSC scaffolds than bone marrow/ ceramics scaffolds. Kadiyala et al.43 also reported that osteogenicity of the MSC was higher than bone marrow.

β‐TCP + Platelet‐Rich Plasma (PRP)

Platelet‐rich plasma contained plenty of platelets and multiple proteins, including platelet‐derived growth factor (PDGF), TGF‐β, vascular endothelial growth factor (VEGF), and epithelial growth factor (EGF). These proteins can promote local neovascularization and absorb multipotent stem cells, monocytes, and apocytes into injured tissues, which is in favor of tissue reparation and regeneration44.

Platelet‐rich plasma is also hard to locally accumulate enough concentration owing to its fluidity, which is necessary for new bone growth. β‐TCP/PRP composite maximize the osteoinductive property of PRP. Batista et al.45 repaired bone defect in the proximal tibias of rabbits with β‐TCP/PRP and β‐TCP/MSC, respectively. The results showed that cortical bone thickness, new bone mass per unit area, bone density, and bone trabecula density were significantly higher in the β‐TCP/PRP group than those in β‐TCP/MSC group. MSC hardly produced equal new bone mass to PRP. In fact, the new bone induced by MSC contained fibrotic tissues among spaces which is hardly replaced by bone tissues. In fact, growth factors secreted by platelets can promote the growth of tissues besides bone, thereby limiting new bone formation. Lobo et al.46 found that the BCP/PRP composite scaffold contained high levels of fatty tissues, whereas single BCP did not contain much new bones. They thought the possible effect of PRP within the body was to maintain or increase the fatty content, not to promote formation of new bones. Growth factors secreted by platelets can accelerate maturation of bones by promoting new vessel growth. In fact, new vessels in single β‐TCP ceramic materials supply sufficient nutrition required by new bones.

β‐TCP + Plasmatransglutaminase (F XIII)

F XIII is one of the blood coagulation factors, which can promote new bone formation. Becker et al.47 used β‐TCP/F XIII to treat distal tibia defect of sheep. In postoperative week 12, the new bone mass reached 27.2% in the β‐TCP/F XIII soaking group, 17.7% in the β‐TCP/F XIII injecting group, 17.6% in the β‐TCP/bone marrow group, and 9.7% in the β‐TCP/peripheral blood group. In the soaking group, F XIII penetrated into the β‐TCP scaffold, therefore new bones formed extensively.

F XIII activated by thrombin can induce the condensation of fibrin monomers and formation of stable blood clot. This is the most important step for osteoblast aggregation. In terms of the cellular view, F XIII could promote the mitosis of osteoblasts and osteoclasts and cell adhesion to β‐TCP scaffold, stimulate the activity of alkaline phosphatase, and stabilize the cell membrane; therefore, it is quite significant in the healing process48.

β‐TCP + Poly‐Caprolactone (PCL)

Poly‐caprolactone is an osteoconductive material, providing excellent mechanical property and biological absorption. Nevertheless, PCL is hydrophobous, which blocks the adhesion and proliferation of various cells, thereby limiting the growth of new tissues49. In contrast, β‐TCP showed favorable hydrophilicity, therefore, the β‐TCP/PCL composite was created to pursue better property. According to a study conducted by Kaewsichan et al.50, flexural modulus and mechanical strength of β‐TCP/PCL vary slightly with increased porosity: 19.8 MPa and 0.62 MPa in porosity of 70%, whereas 15.3 MPa and 0.67 MPa in porosity of 52%, respectively. As for β‐TCP alone, porosity is inversely correlated with the mechanical strength. The porosity of β‐TCP could promote new bone formation, while decrease the mechanical strength. This relates with a higher probability of postoperative scaffold collapse or inner breakage51. In the β‐TCP/PCL compound, calcium and phosphate ions of β‐TCP integrate ‐C = O of PCL for forming stable electrostatic force50, 52. This enhances the mechanical strength of scaffold. Therefore, β‐TCP/PCL composite fulfills the requirements for new bone formation and simultaneously enhances the mechanical strength.

β‐TCP + Metal Ions

Silicated calcium‐phosphate (Si‐Cap) is similar to the structure of bones. It is manufactured by replacing phosphate ions with silicate ions. Spaces in the Si‐CaP composite provide scaffolds for proliferating and differentiating stem cells. To increase local stability, Si released from the matrix promotes chemical bonding between the bone and scaffold. The compound can also upregulate the proliferation and differentiation of osteoclasts53, 54, 55. It promotes the expressions of osteoinductive genes56, 57 and increases the synthesis of type‐1 collagen54, thereby accelerating the new bone formation. In vivo studies showed that Si‐CaP played a pivotal role in repairing bone defect models of sheep and rabbits. Wheeler et al. implanted Si‐CaP or autograft bone between transverse processes and into the isthmus. It was found that both groups did not differ significantly in the fusion rate, stability, motion range, and histological characteristics, such as new bone remodeling, osteoclasts, osteoblasts, and inflammatory reactions. However, in the postoperative 6th month, an increasing trend of cell activity was observed in the Si‐CaP group. This could not be observed in the autograft bone group. Therefore, it can be inferred that Si‐CaP is the most effective alternative of autogenic bone, which has effects of osteoconductivity and osteoinduction and can prevent the complications associated with the bone‐taking area.

In the Zn‐β‐TCP composite, Zn²⁺ either promotes osteoclast apoptosis or attenuates osteoclast activity. It also can decrease absorption of the β‐TCP, which not only prevents local collapse or fracture but also provides favorable conditions for new bone formation. Yasutaka et al.58 found that Zn²⁺ was positively correlated with apoptosis of osteoclasts. In addition, Zn²⁺ could inhibit the activities of carbonic anhydraseII (CAII) and catepsin K/OC2. Both of them play important roles in the degradation of organic and inorganic substances of bone tissues59. Zn²⁺ also can inhibit adhesion of osteoclasts to the β‐TCP scaffold, which blocks early and much greater absorption of β‐TCP through osteoclasts.

The major property of β‐TCP is osteoconductivity which is one of the major causes for β‐TCP and can be used as a bone repairing material. However, only osteoconductivity is devoid of enough bone regeneration and remodeling. Tan et al.60 think that new bone growth require the following factors: (i) bioactive materials with scaffold function; (ii) various growth factors inducing cell differentiation; and (iii) osteoblasts or stem cells having the potential of differentiating into the bone tissue. Therefore, the bone‐repairing effect of β‐TCP can be maximally improved, if we provide β‐TCP with the osteoblasts and/or growth factors for stimulating osteoblast differentiation.

In clinical application, degradation and mechanical property are important physical parameters of β‐TCP, which is mainly associated with the porosity and surface size6, 61, 62. Continuous degradation of β‐TCP provides enough spaces for cell growth, while excellent mechanical properties can maintain the space structure for long‐term cell growth. However, they restrict each other, because more porosity will lead to higher degradation61, 62, 63, 64 and poorer mechanical properties. Hence, to improve physical properties of β‐TCP, it is necessary to combine β‐TCP with other bone repairing materials.

To sum up, β‐TCP composite fully use excellent properties of other bone repairing materials, such as biodegradability, osteoinductivity, osteogenicity and osteoconductivity. These materials make up for the deficiencies of single β‐TCP and endow β‐TCP with more biological and physical properties.