**4.1 Bone graft biocomposites**

The bone graft was prepared as follow: A solution of chitosan (1,7%) (DDA 83% ± 3%, supplied by Biomolecules and Organic Synthesis Laboratory, Ben M'Sick University, Casablanca) was prepared in diluted chloric acid solution (0.2 N). The chitosan was dissolved under ultrasonic agitation. Disodium glycerophosphate solution (0.5 N) was added slowly under agitation at low temperature. The pH was maintained between (6.5–7). The chitosan solutions were then autoclaved. Porous Biphasic calcium phosphate bioceramics (BCP) (50%Beta TCP-50%HA, porosity = 76%, Biomatcan) with average granules size of 135 microns was added slowly and gently homogenized. It was found during the preliminary tests, that the best formulation that preserve homogeneity and injectability have a ratio of BCP comprising between 35% and 50%. Low concentration led to aggregation of the granules and high concentration affects the injectability and the structural stability of the biocomposites. The obtained products were kept at cold temperature till use. The mechanical properties of the obtained biocomposites were measured at physiological temperature (37 <sup>o</sup> C) with rheometer (Brookfield DV3T). The obtained results are reported in the table and figures bellow (**Table 6**, **Figures 7** and **8**).

In this case we notice that the increases of the BCP mass in the chitosan solution increase the mechanical properties of mixture. This increase is not linear. The maximum is obtained for L/S = 40%. Over this limit the biocomposite is less injectable and less elastic. 0.4% of BCP represent the maximum load for this formulation with optimal mechanical properties.


**Table 6.**

*Eleastic moduls of biocomposite bone graft with different BCP.*

**87**

*Chitosan Based Biocomposites for Hard Tissue Engineering*

**4.2 Injectable bone substitute material- biocements**

*Representative example of rheological test obtained at 37°C.*

made by two different chitosan solutions.

*4.2.1* Self-hardening *biocomposites*

**Figure 8.**

biocomposites was prepared as follow:

(MPa) for different ratio L/S is reported in **Table 7**.

*4.2.2* Self hardening *CaP biocements*

The biocements are made by mixing solid (S) and liquid phases (L) they are known to harden in certain conditions, the mechanical properties depend on the solid and liquid compositions. They are used in bone augmentation situations like joint fixation, maxillofacial surgeries, and others. We have tested biocomposites

These materials are made out of a grafted chitosan mixed with Alpha PTC bioceramics fine powder. The biocomposites has the advantage that when it is mixed with the CS solution it forms an injectable paste that turns to rubber-like material. It should provide a good initial mechanical stability for the bone defect and the implant. The hardening of the biocomposites occurs progressively over time. The

Grafted chitosan solution: a mPEG-grafted-chitosan [49] transparent and homogeneous gel was prepared from a liquid chitosan aqueous solution (chitosan 2.0% w/v, pH < 6) and Monomethoxypolyethyleneglycol-N-hydroxysuccinimidylsuccinate (mPEG-suc- NHS). The obtained polymer solution was mixed with fine powder CaP ceramic powder (PTC alpha, Ca/P = 1.50, D50 = 4microns, Biomatcan). The Liquid/ powder ratio (L/S) varies from 0.4, to 0.6. The biocomposites cement pastes were injected in a rubber made cylindrical molds (6 mm in diameter x 12 mm height). The elastic silicone-like articles were demolded and stored at 37°C in humid atmosphere for 24 h to harden. The solid blocs were matured in Simulated Body Fluid (SBF) solution at 37°C for 3, and 7 days. Then washed with cold distilled water and dried at 40oC for 24 h. The obtained biocomposites articles were mechanically tested (Zwick Z010 mechanical testing machine, with a crosshead speed of 1 mm/min). 10 specimens were tested for each test formulation. The measured compressive strength

The biocements are made with crosslinked CS formulations and without chitosan solution were prepared and compared side by side. Chitosan (83% ± 3 DDA)

*DOI: http://dx.doi.org/10.5772/intechopen.98468*

**Figure 7.** *Elastic modulus of biocomposites formulations (KPa, 37°C).*

*Chitosan Based Biocomposites for Hard Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.98468*

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

mechanical properties were evaluated in both of cases.

**4.1 Bone graft biocomposites**

figures bellow (**Table 6**, **Figures 7** and **8**).

*Eleastic moduls of biocomposite bone graft with different BCP.*

*Elastic modulus of biocomposites formulations (KPa, 37°C).*

optimal mechanical properties.

An injectable bone graft formulation and hardening injectable bone cements. The

The bone graft was prepared as follow: A solution of chitosan (1,7%) (DDA 83% ± 3%, supplied by Biomolecules and Organic Synthesis Laboratory, Ben M'Sick University, Casablanca) was prepared in diluted chloric acid solution (0.2 N). The chitosan was dissolved under ultrasonic agitation. Disodium glycerophosphate solution (0.5 N) was added slowly under agitation at low temperature. The pH was maintained between (6.5–7). The chitosan solutions were then autoclaved. Porous Biphasic calcium phosphate bioceramics (BCP) (50%Beta TCP-50%HA, porosity = 76%, Biomatcan) with average granules size of 135 microns was added slowly and gently homogenized. It was found during the preliminary tests, that the best formulation that preserve homogeneity and injectability have a ratio of BCP comprising between 35% and 50%. Low concentration led to aggregation of the granules and high concentration affects the injectability and the structural stability of the biocomposites. The obtained products were kept at cold temperature till use. The mechanical properties of the

obtained biocomposites were measured at physiological temperature (37 <sup>o</sup>

rheometer (Brookfield DV3T). The obtained results are reported in the table and

In this case we notice that the increases of the BCP mass in the chitosan solution increase the mechanical properties of mixture. This increase is not linear. The maximum is obtained for L/S = 40%. Over this limit the biocomposite is less injectable and less elastic. 0.4% of BCP represent the maximum load for this formulation with

**BCP (%) 0 0.36 0.40 0.44 0.5** Chitosan solution (%) 1.7 1.7 1.7 1.7 1.7 Elastic modulus (Kpa) 1.8 3.8 14.2 5.2 2.8 Time (min) 27 115 40 63 62

C) with

**86**

**Figure 7.**

**Table 6.**

**Figure 8.** *Representative example of rheological test obtained at 37°C.*

### **4.2 Injectable bone substitute material- biocements**

The biocements are made by mixing solid (S) and liquid phases (L) they are known to harden in certain conditions, the mechanical properties depend on the solid and liquid compositions. They are used in bone augmentation situations like joint fixation, maxillofacial surgeries, and others. We have tested biocomposites made by two different chitosan solutions.

### *4.2.1* Self-hardening *biocomposites*

These materials are made out of a grafted chitosan mixed with Alpha PTC bioceramics fine powder. The biocomposites has the advantage that when it is mixed with the CS solution it forms an injectable paste that turns to rubber-like material. It should provide a good initial mechanical stability for the bone defect and the implant. The hardening of the biocomposites occurs progressively over time. The biocomposites was prepared as follow:

Grafted chitosan solution: a mPEG-grafted-chitosan [49] transparent and homogeneous gel was prepared from a liquid chitosan aqueous solution (chitosan 2.0% w/v, pH < 6) and Monomethoxypolyethyleneglycol-N-hydroxysuccinimidylsuccinate (mPEG-suc- NHS). The obtained polymer solution was mixed with fine powder CaP ceramic powder (PTC alpha, Ca/P = 1.50, D50 = 4microns, Biomatcan). The Liquid/ powder ratio (L/S) varies from 0.4, to 0.6. The biocomposites cement pastes were injected in a rubber made cylindrical molds (6 mm in diameter x 12 mm height). The elastic silicone-like articles were demolded and stored at 37°C in humid atmosphere for 24 h to harden. The solid blocs were matured in Simulated Body Fluid (SBF) solution at 37°C for 3, and 7 days. Then washed with cold distilled water and dried at 40oC for 24 h. The obtained biocomposites articles were mechanically tested (Zwick Z010 mechanical testing machine, with a crosshead speed of 1 mm/min). 10 specimens were tested for each test formulation. The measured compressive strength (MPa) for different ratio L/S is reported in **Table 7**.

#### *4.2.2* Self hardening *CaP biocements*

The biocements are made with crosslinked CS formulations and without chitosan solution were prepared and compared side by side. Chitosan (83% ± 3 DDA)


**Table 7.**

*Compressive strength (MPa) obtained for different bone cement with modified chitosan solution after 3 and 7 days of maturation (Ref = PTC alpha with water only, L/S = 0.5).*


#### **Table 8.**

*Compressive strength comparison of biocement formulations prepared with water vs. 1 of chitosan solution.*

was dissolved in 1%HCl). The pH was maintained 6.7 to 7 with Sodium glycerophosphate (Sigma Aldrich). The solid phases were selected from different sources of CaP material. The tricalcium alpha (alpha)TCP and Hydroxyapatite (HA) supplied by Biomatcan, tetra calcium phosphate (TTCP, Cambioceramics, NL), Brushite (DCPD) and monocalcium phosphate (MCPM) from Sigma-Aldrich.

The biocomposites were prepared by mixing powder and solutions with predetermined ratio L/S. The paste was handled as mentioned before. When the cements harden, the cylindrical blocs were put in phosphate buffer saline solution at 37°C, pH 7.4 for 24 hours, then washed with cold water and dried at 40°C for 24 hours. The formulations and obtained results are summarized in **Table 8**.

The results of the mechanical tests on both formulations show that the addition of mPEG- grafted-chitosan solution or crosslinked chitosan solution decreases dramatically the mechanical properties of self-herding biocements. It could be explained by the effect of chitosan on the CaP crystal growth during maturation of the biocements, or by the heterogenous structure of the biocements, where chitosan polymer creates some discontinuity in the physical structure. Moreover, the shrinkage of the chitosan network during the drying process could induce a distortion of the article volume thus reducing its mechanical properties. In-vivo testing would be the best approach to assess the mechanical properties of such formulations.

## **5. Conclusion**

In conclusion we have presented some works done related to the development of chitosan, CaP biomaterials that mimic the composition of natural bone. Despite the proven biological benefits and the huge number of research, publications and patents done on the use of chitosan in medical field and especially in hard tissues replacement, there is a big discrepancy between research, commercial and market reality. Less than handful products are marketed mainly for cartilage repair.

**89**

**Author details**

Fouad Dabbarh1

Casablanca, Morocco

\*, Noureddin Elbakali-Kassimi2

solution in bone tissue regeneration for the bioindustry players.

1 Biomatcan Ltd, Fredericton, Nouveau-Brunswick, Canada

2 Department of Chemistry, University of New Brunswick,

\*Address all correspondence to: fdabbarh@biomatcan.com

3 Faculty of Sciences Ben M'Sick, Laboratory of Biomolecules and Organic Synthesis, (BIOSYNTHO), Department of Chemistry, University Hassan II of

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Fredericton, New Brunswick, Canada

provided the original work is properly cited.

and Mohammed Berrada3

*Chitosan Based Biocomposites for Hard Tissue Engineering*

The principal obstacles are proper to the material itself and processing. No validated manufacturing process, variability in the raw material, the formulations developed up to date have low mechanical properties, regulatory burden associated with the endotoxin content that require additional steps and control in the manufacturing process, the sterilization that affect the polymer, the storage, shelf life and stability conditions especially for the liquid and gel formulations. However, some new technologies have been tested to solve some of these problems, such plasma sterilization that delivers free endotoxin chitosan raw material [56]. It is still at early stage and need to be validated technically and economically at large scale. Other improvements still have to come before chitosan and derivative become attractive

*DOI: http://dx.doi.org/10.5772/intechopen.98468*

### *Chitosan Based Biocomposites for Hard Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.98468*

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

**(ml/g)**

*7 days of maturation (Ref = PTC alpha with water only, L/S = 0.5).*

**Formulations Ca/P L/S** 

αTCP-HA-MCPM

TTCP-DCPA-MCPM

**Table 8.**

**Table 7.**

was dissolved in 1%HCl). The pH was maintained 6.7 to 7 with Sodium glycerophosphate (Sigma Aldrich). The solid phases were selected from different sources of CaP material. The tricalcium alpha (alpha)TCP and Hydroxyapatite (HA) supplied by Biomatcan, tetra calcium phosphate (TTCP, Cambioceramics, NL), Brushite (DCPD) and monocalcium phosphate (MCPM) from Sigma-Aldrich. The biocomposites were prepared by mixing powder and solutions with predetermined ratio L/S. The paste was handled as mentioned before. When the cements harden, the cylindrical blocs were put in phosphate buffer saline solution at 37°C, pH 7.4 for 24 hours, then washed with cold water and dried at 40°C for 24 hours.

*Compressive strength comparison of biocement formulations prepared with water vs. 1 of chitosan solution.*

**Compressive strength (Mpa)**

1.55 0.46 12.7 + 3.9 11.2 + 1.5 11%

**Ref 0.4 ml/g 0.5 ml/g 0.6 ml/g**

1.50 0.60 6.8 + 2.5 2.2+ 0.5 67%

αTCP-DCPA 1.33 0.5 17.3 + 3.1 7.9 + 2.2 54% αTCP-MCPM 1.37 0.72 12.8 + 3.9 11.8 + 1.6 7% αTCP-HA 1.52 0.5 29.0 + 4.9 11.3 + 4.8 61%

3 days 23.22 + 3.58 8.51 + 1.76 7.73 + 1.95 5.51 + 1.30 7 days 29.68 + 4.23 9.82 + 0.26 5.69 + 0.94 4.04 + 1.66

*Compressive strength (MPa) obtained for different bone cement with modified chitosan solution after 3 and* 

TTCP-MCPM 1.66 0.55 8.3 + 1.0 2.9 + 0.4 65%

**Compressive strength (Mpa) (1% chitosan solution)**

**Variation (%)**

The results of the mechanical tests on both formulations show that the addition

of mPEG- grafted-chitosan solution or crosslinked chitosan solution decreases dramatically the mechanical properties of self-herding biocements. It could be explained by the effect of chitosan on the CaP crystal growth during maturation of the biocements, or by the heterogenous structure of the biocements, where chitosan polymer creates some discontinuity in the physical structure. Moreover, the shrinkage of the chitosan network during the drying process could induce a distortion of the article volume thus reducing its mechanical properties. In-vivo testing would be

the best approach to assess the mechanical properties of such formulations.

In conclusion we have presented some works done related to the development of chitosan, CaP biomaterials that mimic the composition of natural bone. Despite the proven biological benefits and the huge number of research, publications and patents done on the use of chitosan in medical field and especially in hard tissues replacement, there is a big discrepancy between research, commercial and market reality. Less than handful products are marketed mainly for cartilage repair.

The formulations and obtained results are summarized in **Table 8**.

**88**

**5. Conclusion**

The principal obstacles are proper to the material itself and processing. No validated manufacturing process, variability in the raw material, the formulations developed up to date have low mechanical properties, regulatory burden associated with the endotoxin content that require additional steps and control in the manufacturing process, the sterilization that affect the polymer, the storage, shelf life and stability conditions especially for the liquid and gel formulations. However, some new technologies have been tested to solve some of these problems, such plasma sterilization that delivers free endotoxin chitosan raw material [56]. It is still at early stage and need to be validated technically and economically at large scale. Other improvements still have to come before chitosan and derivative become attractive solution in bone tissue regeneration for the bioindustry players.
