β-CPP bioceramics in alginate 3D Scaffolds as a new material for mineralized tissue regeneration

doi:10.21203/rs.3.rs-4136796/v1

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β-CPP bioceramics in alginate 3D Scaffolds as a new material for mineralized tissue regeneration

https://doi.org/10.21203/rs.3.rs-4136796/v1

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In the pursuit of tailored properties for applied bioceramics in bone graft applications, a personalized mixture of precursor base materials is crucial. Hydroxyapatite (HA), beta tricalcium phosphate (β-TCP), and versatile biopolymers serve as base materials to produce personalized mixtures, each presenting its own set of advantages and disadvantages. Combining these materials addresses limitations of individual components, but further improvements require exploring alternative base materials with distinct properties. This study introduces beta calcium pyrophosphate (β-CPP) as a valuable addition to the base materials, exhibiting intermediate biodegradation properties. When combined with biopolymer alginate, β-CPP enables the fabrication of personalized porous 3D scaffolds. Despite β-CPP being an unwanted by-product in mineralized tissue regeneration, this research demonstrates its innovative potential in stimulating cell interaction within porous 3D structures. The β-CPP/Alginate 3D Scaffold, with a 5:3 w/w ratio, significantly enhances mineralization activity compared to the control. This novel composite, showcasing interconnected cells throughout the 3D structure, presents a promising avenue for mineralized tissue regeneration. The study underscores the importance of optimizing both biodegradation and mechanical features in new bioceramics, highlighting β-CPP as a noteworthy candidate with potential commercial implications.

Physical sciences/Materials science/Biomaterials

Physical sciences/Materials science/Biomaterials/Biomedical materials

Physical sciences/Materials science/Biomaterials/Biomineralization

Humanity has dealt with bone fractures since ancient times, however, several circumstances, including longer life expectancy, more exposure to accidents, knowledge of the various bone disorders that can affect the body, and the severity of the damage, can lengthen the period that the fractured region calcifies ^1,2. Thus, the development of materials used in dentistry and orthopedics depends critically on the hunt for innovative technologies that promote more effective healing and regeneration ³.

Calcium phosphates bioceramics, such as hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP), dominate the lucrative bone graft market in different ways, such as injectable, in pieces, in cylindrical, block and 3D Scaffolds shapes ^4–6. HA (Ca/P ratio = 1.67) is widely used in biomedical applications due to its structural and chemical similarities to biological calcium phosphates. However, HA lacks certain ions and can be substituted by carbonate ions ⁷. β-TCP (Ca/P ratio = 1.5) is more soluble than HA, making it a popular biomaterial for bone tissue applications. Fine-tuning the HA/βTCP ratio enhances resorption rates and modifies biological characteristics during bone regeneration ⁸.

Many studies have been conducted on HA and β-TCP-based composites about material selection, meanwhile, beta calcium pyrophosphate (β-CPP, Ca/P ratio = 1) is presented in literature as an intermediate biodegradation property product ⁹. The β-CPP is a by-product of β-TCP synthesis since trace levels of β-CPP affect the phase transition between α-TCP and β-TCP, resulting in a certain calcium deficiency in these materials. ^10,11. Faced with the biological environment, Santonini et al found that the presence of 1% of β-CPP reduces the in vitro β-TCP resorption rate by 40% ¹². Lin et al noticed an increase in inflammation surrounding β-TCP implants when the concentration of calcium pyrophosphate was raised ¹³. On the other hand, in the absence of additional calcium phosphate phases, β-CPP demonstrated superior osteoconductive ability and biodegradation over porous HA implants, making it a more appealing option for bone grafting ¹⁴.

However, potential new bioceramics not only require optimized biodegradation but also mechanical features ^15,16. Achieving this often involves the interaction of bioceramics with other materials to enhance their viability in mineralized tissue ^17–21. The extracellular matrix (ECM) plays a crucial role in providing physical scaffolding and regulating various cellular functions ²². Natural biomaterials like agarose, collagen, gelatin, chitosan, carrageenan, and fibrin serve as successful substitutes for the ECM ²³. Sodium alginate, a pH-responsive, biodegradable, and biocompatible polysaccharide derived from seaweed, is a notable choice ²⁴. Composed of mannuronic acid (M) and guluronic acid (G) units, sodium alginate forms gels in the presence of bivalent cations like calcium ions (Ca²⁺) ^25,26. This versatile polymer finds widespread usage in tissue engineering applications such as scaffolding, cell encapsulation, and drug/growth factor deposition ^27–32.

Herein, to demonstrate a real impact in this area, β-CPP was combined with sodium alginate to manufacture 3D Scaffolds for mimicking bone tissue structures. In addition, the challenges associated with the choice of an effective alginate matrix and the response to the cell adhesion and spreading through these 3D Scaffolds were discussed. To the best of our knowledge, this makes β-CPP an innovative new based composite for mineralized tissue regeneration, able to widen the ability to tailor the biodegradation in a new potential biomaterial to be marketed.

2.1 Synthesis of β-CPP ceramics

Using a neutralization reaction, an 0.5 L of phosphoric acid solution (H₃PO₄, 0.8 mol L^− 1) was slowly dripped, over 3h, into 0.5 L of calcium hydroxide solution (Ca(OH)₂ 0.6 mol L^− 1) under mechanical stirring (350 rpm), similar to previous work ⁷. After this reaction period, the sample was left to rest for 2h and then vacuum filtered and freeze-dried. The samples were then subjected to different sintering temperatures (600, 750, 900 and 1150ºC) during 3h.

2.2 Obtaining of β-CPP/Alginate 3D Scaffolds by freeze-drying method.

The 3D Scaffolds prepared using the freeze-drying technique were manufactured using two types of alginates, high and low viscosity (denoted by the letters A and B, respectively). The same preparation and crosslinking procedure were used for both polymeric matrices, as well as the same mass compositions of β-CPP. 100 mL of Alginate suspension was prepared at 3% (w/w), before of β-CPP addition. The system was maintained under constant stirring at 250 rpm for 30 minutes until the polymer matrix was completely dissolved. Four groups of each type of alginate were obtained, as presented in Table 1. The composites were placed in a plastic petri dish and frozen in an ultrafreezer at -80ºC for 24h, to then be freeze-dried for 4 days. After drying, each scaffold was added to a solution containing 3% (w/v) of calcium chloride (CaCl₂), for 3h, to ensure an efficient ionic crosslinking of the sodium alginate, as already established in other works ^33,34. Then, the cross-linked biomaterial was frozen once again for 24h in the ultrafreezer at -80ºC and subsequently freeze-dried.

Table 1

3D Scaffolds composition.
3D Scaffold	Composition
AlgA	alginate solution
AlgB	alginate solution
β-CPP/AlgA/1/1	1:1 ratio of of β-CPP and alginate solution
β-CPP/AlgB/1/1	1:1 ratio of of β-CPP and alginate solution
β-CPP/AlgA/5/3	5:3 ratio of of β-CPP and alginate solution
β-CPP/AlgB/5/3	5:3 ratio of of β-CPP and alginate solution
β-CPP/AlgA/2/1	2:1 ratio of of β-CPP and alginate solution
β-CPP/AlgB/2/1	2:1 ratio of of β-CPP and alginate solution

2.3 Physicochemical characterization

Furthermore, the following physicochemical characterizations were carried out (more details in the SI): Fourier-transform infrared and RAMAN spectroscopy (FTIR and RAMAN), X-ray diffraction analysis (XRD), Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), thermogravimetry, mechanical, compression tests, swelling and degradation tests. The indexing of the samples was performed by the Crystallographica Search-Match software and the β-CPP and HA ratio were made by comparison between the amorphous and crystalline region present in 10 to 60 2θ ³⁵.

2.4 Response of osteoblastic lineage cells cultured into β-CPP/alginate 3D Scaffolds.

The study employed pre-osteoblastic MC3T3-E1 cells for in vitro tests, utilizing a high viscosity β-CPP/Alginate 3D Scaffold. Cells were cultured in complete α-MEM medium with fetal bovine serum (FBS), penicillin, streptomycin, glutamine, and amphotericin B. The 3D scaffolds were standardized and disinfected before cell seeding. Cell viability and proliferation were assessed using alamarBlue reagent and fluorescence microscopy at intervals of 1, 7, and 14 days. The biomineralization capacity of the scaffolds was evaluated after 14 days in an osteogenic differentiation medium ^36,37. More detailed in vitro methodology can be found in the supplementary information.

2.5 Statistical analyses

Fluorescence images were analyzed descriptively. The remaining data were inferentially analyzed using SPSS version 26.0 software (IBM Inc., Chicago, IL, USA). For each set of data, the sampling distribution (Shapiro-Wilk) and homogeneity of variances (Levene) were evaluated. For the model with repeated measures (cell viability), sphericity was assessed using the Mauchly test and multiple comparisons were calculated using the Sidak test. For one-way ANOVA (mineralized matrix formation), the Welch method was applied to correct unequal variances and multiple comparisons calculated with the Games-Howell test. All inferences were made considering a pre-established level of significance of 5%.

3.1 β-CPP characterization

In the diffractograms present in Fig. 1 it was observed that with the increase in sintering temperature there was a gradual conversion of the ceramic precursor into a more stable calcium phosphate phase. It is expressed in Table 2 that the new phases formation in the sintered samples refer to the formation of HA and the β-CPP phases. However, different proportions of each of the chemical species are found, depending on the sintering temperature used.

Due to the presence of a rich [PO₄³⁻] ion environment, with the increase in the sintering temperature in the precursor there is also an increase in the percentage of β-CPP ¹⁴. As these temperatures increase, the diffractograms begin to become more defined and closer to the main peaks found in the pattern used. The sinterization at 1150°C for 3h allowed the formation of β-CPP phase, with more than 97% purity. Higher sintering temperatures were not used, due to their influence on the formation of the α-CPP phase, which begins to be formed from these temperature conditions ^38,39. FTIR spectra (Fig. 1), has bands at 500, 550, 900 and 970 cm^− 1 in all the sintered samples, attributed to ions PO₄^{3– 7}; in the region of 1000 to 1200 cm^− 1, bands attributed to P = O stretching were observed and bands found in 715 cm^− 1, characteristic of the symmetric P-O-P mode, found in pyrophosphates ⁴⁰. All identified bands confirm the presence of the main functional groups present in β-CPP ceramics.

Raman spectra also found in the Fig. 1, shows the presence of bands related to the doubly and triply degenerate stretching mode of the group PO₄³⁻ (450–590 cm^− 1) were observed, as well as the symmetric and asymmetric stretching mode of the group PO₄³⁻(in 960 and 990 cm^− 1 and in the range 1060–1080 cm^− 1, respectively) ⁴¹. However, the presence of a band at 745 cm^− 1 was also observed in both comparative spectra, which became more evident with the increase in sintering temperature. As already proven in the literature, the increasing formation of the β-CPP phase during sintering also induces a percentage increase in the P₂O₇ species. With its increase, the presence of O-P-O bonds, attributed to 745 and 1045 cm^− 1, also increased, becoming more evident in samples with greater amounts of β-CPP in their composition ⁴².

The EDS technique was used to determine the Ca/P ratio of each of the sintered samples, also described in Table 2. At lower sintering temperatures, Ca/P ratios are seen closer to the theoretical value found in the HA phase (Ca/P = 1.67). With its a gradual conversion of the precursor into more thermally stable phases, it is observed that only at high temperatures the Ca/P ratio starts to be like the theoretical value of β-CPP phase (Ca/P = 1.0). This occurred because only under these drastic reaction conditions will occur the condensation of hydrogen phosphate groups in a more effectively, allowing the β-CPP phase to be formed ⁴³.

Table 2

Percentage of HA and β-CPP phases ceramic samples sintered at different temperatures.
Sintering temperature	Phase		Ca/P ratio
Sintering temperature	HA (%)	β-CPP (%)	Ca/P ratio
600°C	44	17	1.27
750°C	7	89	1.18
900°C	1	96	1.12
1150°C	< 1	97	1.08

Considering that the application of these bioceramics will be aimed at bone replacement, their high degree of purity and well-defined physicochemical properties are essential to ensure not only an increase in the adhesion, proliferation, and integration of osteoblastic cells, but also to prevent the occurrence of infections, inflammations, and adverse allergic reactions in the patient's body. Among the sintered groups, the sintered samples at 1150ºC (presenting the β-CPP phase with a purity higher than 97%) were considered the most promising biomaterial to carry on the association with a polymeric matrix of alginate.

3.2 β-CPP/Alginate 3D Scaffolds

By analyzing the thermograms found in Fig. 2, a comparison was made of the different 3D Scaffolds using the high and the low viscosity alginate matrices. A loss of mass was observed for all the 3D Scaffolds in both groups, in the temperature range of 30 to 250°C, related to the evaporation of water from the polymeric structure. Although the samples were dry before testing, sodium alginate is a hydroscopic compound, which in turn may have absorbed water molecules from the air before the analysis was carried out ⁴⁴.

Another loss of mass was found in these 3D Scaffolds occurred at 250 to 350ºC, attributed to the complex degradation process of polysaccharides ⁴⁵. From 350ºC to near to 650ºC, a last stage of decomposition was observed, resulting from the decomposition of sodium carbonate ⁴⁶. Comparing the main groups, greater thermal stability was found with the gradual increase in the amount of β-CPP in the composition, attributed to its high intrinsic thermal stability and strong particle interaction in the alginate matrix ⁴⁷.

Considering the proportions of β-CPP for the alginate matrix of 1:1, 5:3 and 2:1, it was expected a residual mass values corresponding to the residual value found for the pure matrix plus the respective proportion of bioceramics added. Thus, a residual mass near to 23% were found for both standard 3D Scaffolds samples (AlgA and AlgB) consistent with the literature ⁴⁸. With the addition of β-CPP it was expected a residual percentage being like 46% for the 1:1 ratio, 61% for 5:3 and 69% for 2:1. Nevertheless, the experimental data was different from the expected, as described with more details in the supplementary information.

For the 3D Scaffolds prepared with the high viscosity alginate matrix it was observed a similarity of theoretical and practical residual values. This is related to the high homogeneity quality achieved during the manufacture of these alginate hydrogels containing β-CPP, presenting a reliable and known composition. However, the theoretical and practical residual masses do not coincide for the 3D Scaffolds made with the low viscosity alginate. It leads to the hypothesis that part of the low viscosity alginate matrix was possibly degraded by the reaction medium during its mixing with the β-CPP bioceramics. As a result, the quantity of β-CPP particles became greater, resulting in the formation of a composite with different component proportions expected.

Morphological and comparative analyzes of the two groups of β-CPP/Alginate 3D Scaffolds, presented in Fig. 3, were carried out using SEM technique. A high pore interconnectivity was observed in the standard 3D Scaffolds (AlgA and AlgB), a fundamental property for an effective vascularization and nutrient transport process, as reported in the literature ⁴⁹. Using a higher magnification, agglomerates linked to the alginate polymeric structure were seen in both groups with a 1:1 concentration, possibly due to the presence of β-CPP bioceramics. For the groups with concentration 5:3 and 2:1, a densification of the polymeric structures of the continuous phase was observed.

With this gradual increase in the concentration of the β-CPP bioceramics, a less smooth and homogeneous distribution throughout the polymer matrix was observed. Still, it can be confirmed that β-CPP bioceramics were successfully incorporated into hydrogels based on different alginates for manufacturing 3D Scaffolds. And the emergence of agglomerates has inevitably occurred due to the nano and micro scale of these bioceramic particles ⁵⁰.

Swelling tests were carried out for a period of 2 days with both alginate matrices samples, Fig. 4. In high viscosity β-CPP/Alginate 3D Scaffolds were observed an average mass gain of 500% for all groups in the first three hours of analysis, due to the hydration of the hygroscopic alginate structures. During this period the 3D Scaffold AlgA presented the highest hydration rate of the groups, since it does not contain β-CPP bioceramics, which is almost insoluble in water, presenting around 510% when compared to its initial mass. For the β-CPP/AlgA/1/1 and β-CPP/AlgA/2/1 composites there was a very similar hydration stabilization, close to 490% and around 550% for the β-CPP/AlgA/5/3 group.

The group formed from the low viscosity matrix also had a more pronounced hydration in the first 3h of analysis (Fig. 4). For the 3D Scaffold AlgB, there was a constant gain in the degree of swelling when compared to the other groups. This hydration was equivalent to 620% above the initial measured mass. The plateau for the β-CPP/AlgB/1/1 and β-CPP/AlgB/2/1 groups occurred after 24h, with values close to 530% hydration. A higher degree of swelling was observed in the β-CPP/AlgB/5/3 group, but after 24h a loss in water retention capacity stabilized at 555%.

Higher swelling values were found for the 3D Scaffolds using the low viscosity matrix when compared to the high viscosity group. One explanation for this phenomenon is that a lower viscosity can result in a greater spacing between the polymer chains after the freeze-drying process, thus making greater hydration of the 3D Scaffolds possible. It is also known that a modification in the proportions of polyguluronic acid, present in the alginate chains, results in different properties and functionalities, including the degree of liquid retention ⁵¹. Besides that, the water absorption occurred during just this first period of immersion and after 24h the swelling rate was stabilized, and minimal variations were observed for both groups. For tissue engineering applications, the developed 3D Scaffold must have a good interaction with the living cells, ensuring nutrient permeation and in vitro phase growth. In this case, choosing a specific degradation capability is critically important, for an effective hydration potential and tissue repair capacity ⁵².

After 7 days in PBS medium, a high degradation value was seen for all 3D Scaffold groups, especially for the groups without the presence of bioceramics, showed in Fig. 4, resulted by the aqueous medium used, which influenced mechanisms of dissolution and hydrolysis of polysaccharide chains ⁴⁴. For the groups with the high viscosity alginate this degradation was around 65% for the AlgA, due to its hydrophilic nature, which leads to greater water uptake and subsequent degradation. However, this value reached a plateau after this period and remained stable in the subsequent days. Until the 14th day, it was observed that the β-CPP/AlgA/5/3 and β-CPP/AlgA/2/1 3D Scaffolds had the lowest degradability. But, after this period there was a degradation around 10% in the β-CPP/AlgA/5/3 3D Scaffold, with the possible release of β-CPP into the reaction medium. For β-CPP/AlgA/1/1, it was seen that after 21 days around 43% of the initial mass was maintained.

For the low viscosity group a faster degradation rate was found when compared to high viscosity groups. The degradation found was approximately 80% for all groups after reaching the 21st day mark. This effect may be directly related to the amount of empty spaces present in the alginate polymeric structure, which facilitated the scission of the chains and oxidation of the sugars present in the composition. The difference in degradation profiles may also be related to the different molecular masses used. A greater degree of degradation could be attributed to a low molecular weight alginate matrices ⁵³.

The mechanical compression tests were carried out in each group, also described in Fig. 4. For the high viscosity group, a good correspondence between the concentration of β-CPP and the mechanical properties was observed. In these cases, it was found that 3D scaffolds with a 2:1 ratio showed a significantly higher compressive strength than in the other groups, confirming that the compressive properties depended on a high concentration of β-CPP within the porous polymer matrix. This resistance property also provides evidence regarding the effectiveness of the interaction between bioceramic particles within the porous chains in the alginate-based matrix ⁵⁴.

In this group the tension was increased slightly with the corresponding increase in deformation, consistent with the behavior of polymer foam-type composites, according to the literature ⁵⁵. This behavior was confirmed in the graphs based on the existence of two regions, one called a plateau, where the collapse of the plastic structures in the composite began, followed by the second region where the tension increased rapidly due to the effective densification of the foam structure ⁵⁴.

Testing the low viscosity group, the same typical stress-strain behaviors were observed for porous composites. However, in this group, the presence of lower compressive strength could also be assessed, possibly caused by greater porosity of the composite. This compressive strength should in theory be greater with increasing amount of β-CPP, but a much higher strength was observed in the 3D Scaffold AlgB than in the samples with a 1:1 and 5:3 ratio. This may have occurred due to a continuous and dispersed phase interaction not as effective as expected ⁵⁴. In the 3D β-CPP/AlgB/2/1 3D Scaffold, a greater deformation when compared to the AlgB sample and only then the polymer chains densification occurred.

3.3 In vitro tests

As the results obtained from low viscosity alginates 3D Scaffolds, in the previous section, were considered unsatisfactory, the in vitro test was carried out only for the group made with the high viscosity matrix.

Throughout all analysis periods, the fluorescence images obtained by the live/dead assay showed a large and increasing number of viable cells for all formulations in this group of 3D Scaffolds. Since green fluorescence marks viable cells (calcein) and red fluorescence marks cells in the process of death (ethidium homodimer), a great proliferation was highlighted for the 3D Scaffolds containing β-CPP, especially between the 7th and 14th day (Fig. 5A). For all formulations, the identified red fluorescence intensity was visually lower than the green fluorescence, indicating that the quantity of viable cells was greater than those in the process of death. The general cytocompatibility of alginate is already consolidated ^56,57, agreeing with the results found in these fluorescence images.

Thus, the control group can be considered suitable for MC3T3 cell adhesion, however it was seen a higher cell spreading in the samples containing of β-CPP bioceramics, indicating that the presence of β-CPP stimulated greater interaction between the cells and the 3D Scaffold formulations. This similar behavior can also be seen in other works associating polymeric 3D Scaffolds with small amounts of other calcium phosphates in the composition ^58,59.

This effect has been related to properties of porosity, presence of surface charge, promotion of ionic environment and low β-CPP solubility; Together, these properties also exert an important role for cell adhesion, by facilitating the adsorption of adhesive proteins and contributing to the maintenance for a functional structure ^60,61.

Furthermore, the fluorescence images made it possible to observe the distribution of cells throughout the biomaterials. This was observed by the presence of interconnected and diffused cells throughout the interior of the biomaterials, a phenomenon that seemed to be intensified throughout the analysis periods, resulting in larger and confluent foci of adhesion, and spreading.

Figure 5B shows the viability of MC3T3-E1 pre-osteoblasts over 1, 7 and 14 days of cultivation on different formulations of the high viscosity alginate group. Comparing the effects of different samples containing β-CPP with the control (AlgA), it was possible to verify the effect of the evaluation period (p < 0.001), the sample p < 0.001) and the absence of interaction (p = 0.071). Thus, comparing the evaluation periods, there was no increase in viability between 1 and 7 days (p = 0.37), while the later period of 14 days showed an increase compared to the previous ones (p ≤ 0.011). Regarding 3D Scaffold samples, all formulations containing β-CPP resulted in greater viability compared to the control (p ≤ 0.014), however, all were comparable to each other (p ≥ 0.210).

In Fig. 5C is presented the formation of a mineralized matrix of MC3T3-E1 pre-osteoblasts after 14 days of cultivation on these 3D Scaffolds. Two samples containing β-CPP promoted an increase in mineralization activity in relation to the control, being the 5:3 ratio (p < 0.001) which increased around 8 times and 2:1 ratio, which increased around 3 times (p = 0.033). Compared to each other, these two formulations differed in this parameter (p = 0.008). It was possible to confirm properties regarding the effectiveness of osteoinduction and osteoconduction properties of the β-CPP bioceramics, as well as their non-toxicity when in contact with the biological environment.

In the β-CPP/AlgA/5/3 3D Scaffold was observed the highest positive stimulus, consequently caused by the activation of the intracellular signaling pathways. The ions Ca²⁺, in this 3D Scaffold composition, ensured an effective modulation in the cells, cause its differentiation and produce the mineralized matrix ³⁷. After passing this ideal concentration (observed that for the group 2:1 ratio), an inhibitory stimulus occurred, due to a toxic behavior caused by the excessive [Ca²⁺], present in the composition of the 3D Scaffold ⁶². Furthermore, a comparison with other calcium phosphates bioceramics already marketed and in vivo investigations should be focused to provide additional evidence for the efficiency of the intracellular signaling pathways associated with these β-CPP/Alginate 3D Scaffolds.

In summary, we show that β-CPP, an undesirable intermediary by-product, is a valuable bioceramic to the fabrication of 3D Scaffolds. In particular, it is clear the use of a high viscosity alginate allowed the formation of a homogeneous 3D structure, presenting a linear result of mechanical, swelling and degradation behavior with the β-CPP concentration increases. The osteoinductive and osteoconductive properties of this group of 3D Scaffolds could be observed due to excellent cell proliferation and interconnectivity, in response to the osteoblastic lineage. Taken together, all these properties guarantee more flexibility to tailor-made the association of polymeric matrices with other potential calcium phosphates to explore even more the mineralized tissue regeneration applications.

Acknowledgements

This work was developed within the scope of the project PTDC/NAN-MAT/3901/2020 financed by FCT/MCTES, by the Fundo Europeu de Desenvolvimento Regional - FEDER, through do Programa Operacional Competitividade e Internacionalizaçao e do Programa Operacional Regional do Centro, CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national portuguese funds through the FCT/MCTES (PIDDAC) and the Brazilian funding agencies CNPq and DAI.

DATA AVAILABILITY

The data of this study are available on request from the corresponding author.

AUTHOR CONTRIBUTIONS

G.C.P.: conceptualization, methodology, data analysis, and writing the original draft; R.D.P.: data analysis and methodology; I.P.M.S. and C.A.: in vitro tests and data analysis; E.T.C.C.: study conception and design; C.A.S.C., J.H., N.J.O.S. and A.C.G.: conceptualization, editing, and project supervision. All authors read and approved the final manuscript.

INTEREST STATEMENT

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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β-CPP bioceramics in alginate 3D Scaffolds as a new material for mineralized tissue regeneration

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Abstract

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INTRODUCTION

MATERIALS AND METHODS

2.1 Synthesis of β-CPP ceramics

2.3 Physicochemical characterization

2.5 Statistical analyses

RESULTS AND DISCUSSION

3.2 β-CPP/Alginate 3D Scaffolds

3.3 In vitro tests

CONCLUSIONS

DECLARATIONS

REFERENCES

Additional Declarations

Supplementary Files

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