To what extent is hydroxyapatite a suitable material for bone regeneration scaffolds?
Hydroxyapatite is a calcium phosphate mineral with the molecular formula Ca5(PO4)3OH. Hydroxyapatite crystallises to form a ceramic material which forms the mineral phase of bone. Since hydroxyapatite is already present in the human body, making up 65% of all bone, it is valid to assume it would have excellent biocompatibility. Indeed, this is true, as has been confirmed, first, by professor M. Jarcho and many other subsequent research teams. The first instances of osteogenic applications of hydroxyapatite date back to the 1950s when this material, along with other ceramics, was used to fill in bone defects. Nevertheless, it is in recent years that research into hydroxyapatite scaffolds for bone regeneration therapies has accelerated, and much of this research is currently ongoing. So far, there is ubiquitous information on the biocompatibility of hydroxyapatite and the efficiency of different scaffold shapes and chemical compositions, however, little is known about the specific interactions between hydroxyapatite and mesenchymal stem cells, leading to bone regeneration.
With much ongoing research, one can be sure that the answers to the mysteries of the molecular interactions of hydroxyapatite with mesenchymal stem cells will be uncovered very soon. In the meantime, let us focus on what is known about this unique material. At the moment, autografting is the primary method of bone repair. Autografting works by taking a tissue sample from the patient’s own body and transplanting it into the location where regeneration is required. This method, however, has its drawbacks, which include limited tissue availability, donor site morbidity, severe pain, difficulty in producing correctly shaped grafts, and failure rates of up to 50% for some sites. Looking to minimise risks and mitigate these drawbacks, scientists are looking to find a material or combination of materials with suitable properties for creating scaffolds for bone regeneration, which can house stem cells and promote their differentiation. For these demands to be met, a material should provide a physiological environment for endogenous bone cells, and be resorbed at a rate equal to that of new bone tissue formation.
One material which has shown potential is polylactic acid. Polylactic acid is a semicrystalline, biobased polyester, with good biocompatibility and biodegradability. Nevertheless, polylactic acid also has significant shortcomings such as its poor mechanical strength and low cell affinity. Aiming to solve this problem, scientists have turned to the incorporation of ceramics into polylactic acid, or vice versa to overcome its hydrophobicity, strengthen it mechanically, and stimulate osteoinduction and osteointegration of implanted scaffolds.
One such ceramic is hydroxyapatite. Due to its staggering similarity with the mineral portion of bones, it can form strong chemical bonds with living tissue, potentially giving scaffolds optimal osteointegration properties, while also mechanically strengthening polylactic acid. Combining polylactic acid (PLA) and hydroxyapatite (HA) into one would form a PLA/HA composite, in which PLA could provide physical support for cell growth, while HA would enhance cell proliferation and osteoinduction. Moreover, HA has been found to enhance the mechanical properties, fibre diameter and pore size of PLA nanofibers, all of which are beneficial properties for harbouring living cells. The cells of choice to be implanted into such scaffolds are mostly mesenchymal stem cells (MSCs) due to their ability to easily differentiate into osteoblasts. Lastly, it is highly important for the scaffolds to not trigger an immune response and not be rejected by the body, hence monitoring immune cells, and in particular dendritic cells (DCs), is crucial.
In a recent study, researchers looked into the use of PLA/HA scaffolds for bone regeneration and their findings were fascinating.
Firstly, they discovered that the tensile and compressive properties of PLA/HA scaffolds were much better than those of neat PLA scaffolds. Neat PLA scaffolds were tested against two types of PLA/HA scaffolds, composed of 20% and 25% HA, respectively. The scaffolds containing 20% HA showed a 22% increase in tensile elastic modulus, and a 48% increase in compression elastic modulus, when compared to neat PLA scaffolds. In turn, the scaffolds containing 25% HA showed a 26% increase in tensile elastic modulus, and a 15% increase in compression elastic modulus, when compared to neat PLA scaffolds. The slight difference in mechanical properties between the two composites can likely be explained by the different distribution of HA particles. Regardless of the differences, these are significant improvements in the mechanical properties of the scaffolds.
Secondly, the scientists examined the efficiency of the scaffolds’ in vitro degradation. Ideally, the scaffold should degrade at the same rate as new bone tissue forms. The three types of scaffold were tested on a 12 week timescale and all underwent mass loss. The scaffolds with 25% HA exhibited the highest degradation rate, followed by the 20% HA scaffolds, and then by neat PLA scaffolds. This was likely due to the fact that HA improved the scaffolds’ hydrophilicity and increased the scaffolds’ porosity, favouring the absorption of water and accelerating degradation rates. Another important factor which was monitored in this test was the pH around the scaffold. When PLA is hydrolysed, it gives acidic products, mainly lactic acid which lower the solution’s pH. Therefore, one would expect that neat PLA scaffolds would have achieved the lowest pH. Nevertheless, this was not the case as the neat PLA scaffolds tied for lowest pH with 25% HA scaffolds, going down from a pH of 7.4 to approximately 5.3. This may seem surprising as HA should have a neutralisation effect on the lactic acid, and yet the composite with the lowest proportion of PLA and highest proportion of HA tied for lowest pH with neat PLA scaffolds. However, after a little thought this finding becomes much less intriguing as one has to remember the 25% HA scaffolds also had the highest degradation rate, leading to increased acidification. The 20% HA scaffolds, on the other hand, only decreased from a pH of 7.4 to 6.4. From this data, one can conclude that the 20% HA composite is optimal for scaffolds due to its intermediate degradation rate and reduced acidification (over-acidification can be harmful to cells).
Moreover, every type of scaffold was tested for activation of dendritic cells (DCs). This test is highly important to ensure the scaffolds are biocompatible and do not trigger an immune response which could lead to the scaffolds’ rejection by the body. None of the 3 different types of scaffolds showed any activation of DCs, demonstrating their excellent biocompatibility. This is extremely important for a material to be used for in-vitro bone regeneration therapies.
Furthermore, the scaffolds were tested for osteointegrative and osteoinductive properties. Once again, all three scaffold types showed no signs of cytotoxicity to MSCs and MSCs showed efficient adhesion to the scaffolds’ surfaces. Efficient adhesion of MSCs to the scaffolds is important as it provides structure and allows for successful differentiation. Moreover, the scaffolds were tested for alkaline phosphatase levels across 21 days to determine the extent of their osteoinductive properties. Alkaline phosphatase contributes to the initiation of osteoblast mineralization during extracellular matrix synthesis (the second stage of MSC differentiation). During extracellular matrix synthesis, alkaline phosphatase catalyses the hydrolysis of organic phosphate to inorganic phosphate which is a key factor for mineral deposition and bone formation. Hence, alkaline phosphatase levels are a reliable metric for MSC differentiation. PLA/HA scaffolds showed higher alkaline phosphatase activity than neat PLA scaffolds. PLA/HA scaffolds were also shown to efficiently induce osteogenesis, even in the absence of classic osteogenic culture and stimuli. This is an extremely useful property of the composites as it allows the scaffold to induce osteogenesis on its own and reduces the need for osteogenic culture, reducing costs and removing delivery complications. The final stage of MSC differentiation is extracellular matrix mineralization, during which calcium deposits are formed. PLA/HA scaffolds have a strong enhancement effect on extracellular matrix mineralization, which can be explained by the dissolution of HA in calcium and phosphate ions, which play essential roles in bone metabolism. Calcium is known to support cell adhesion, proliferation, differentiation, and extracellular matrix mineralization, while phosphate regulates cell proliferation and stimulates the expression of key proteins involved in the extracellular matrix mineralization process.
Lastly, the impact of the scaffolds on the expression of osteogenic genes in MSCs was examined. The expression of the following 4 genes was tracked; BMP-2, which is a bone morphogenic protein — an essential growth factor for directing MSC differentiation towards osteoblasts, through the SMAD-signalling pathway; RUNX-2, which is a key transcriptional factor for initiating osteogenic differentiation by regulating the expression of major bone matrix genes; COL1A1, which is considered an early osteoblast marker and is expressed during the transformation of of osteoprogenitor cells into preosteoblasts; OCN, which is a calcium-binding protein intrinsic to the organic matrix of bones, expressed in mature osteoblasts. The results obtained from the fluctuations in expression of these 4 genes showed that neat PLA scaffolds do not support the final stages of MSC differentiation, such as extracellular matrix mineralization and OCN expression, and could possibly be better used as osteoconductive scaffolds, providing a suitable substrate for cellular activity. Whereas, PLA/HA scaffolds supported healthy and timely expression levels of all of the 4 monitored genes, supporting the claim that PLA/HA scaffolds efficiently support and promote the osteogenic differentiation of MSCs, even in the absence of classical osteogenic stimuli, thereby showing excellent potential for bone replacement therapies.
In conclusion, hydroxyapatite is a ceramic material with excellent biocompatibility and interesting properties but fairly few applications on its own. However, when combined with another material such as polylactic acid and developed into a composite scaffold, it can efficiently support and promote the osteogenic differentiation of MSCs, even in the absence of classical osteogenic stimuli, thereby showing excellent potential for bone replacement therapies.
References:
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