**4.2 Demineralized bone matrix: Effect of the acid concentration**

Demineralized bone matrix (DBM) is often applied in orthopedics, periodontics, oral and maxillofacial surgery because of its inherent osteoconductive and osteoinductive properties, generally related, as mentioned, to bone morphogenetic proteins (BMPs) (Bauer & Muschler, 2000; Eppley et al., 2005; Katz et al., 2009). In fact, as mineral is removed, the matrix associated BMPs become available rendering DBM grafts osteoinductive (Pietrzak et al., 2009). These grafts can be used either alone (Libin et al., 1975; Morone & Boden, 1998; Pietrzak et al., 2005) or in combination with bone marrow, autogenous bone graft, or other materials (Kim et al., 2002; Kucukkolbasi et al., 2009; Nade & Burwell, 1977). Additionally, DBM exhibits elastic features, being easily shaped to fill osteochondral lesions with different shapes and sizes (Costa et al., 2001).

Despite the extensive use of DBM, conflicting results have been published in the literature regarding its bone-inducing abilities. This may be a consequence of following different

Characterization of Bone and Bone-Based Graft Materials Using FTIR Spectroscopy 333

Fig. 13. FTIR spectra from the outer surface and from the core of the human bone samples after immersion in 1.2 M HCl for different time intervals. Demineralization proceeds from the surface into the core of the bone samples, as evidenced by the absence of phosphate

collagen bands intensity) and progresses to the interior of the samples. These results support the concept of a diffusion model and agree with the proposed theory of the unreacted core

This study was complemented with kinetic profiles and analysis of the samples´ structural modifications. As expected, increasing the acid concentration led to an increase in the demineralization rate, but not in a proportional way. In addition, microscopic observations demonstrated that despite the structural deformation resultant from demineralization, the basic bone microstructure was preserved. The loss of mineral led to a progressive reduction of mechanical strength and an increase of plastic properties (e.g. flexibility and elasticity) of

Although the deterioration of the organic component of bone was not examined in detail in this work, other studies using FTIR to analyse the effect of acids on the composition and structure of collagen during extraction from different tissues, may provide useful information on that subject. In fact, acid treatment of collagen samples was found to originate reduction of intermolecular cross-linking and hydrolysis of peptide bonds, as evidenced after curve-fitting in the spectral regions of the Amide I, II and III bands of collagen (Muyonga et al., 2004). These changes in collagen composition explained the observed loss of structural order. In addition, the amount and characteristics of the extracted fragments of collagen was related with the experimental conditions. These results agree with those from a FTIR study concerning the cross-linking of a collagen-hydroxyapatite nanocomposite with glutaraldehyde, as a model for the bone matrix (Chang & Tanaka, 2002). The spectral analysis showed that the increase of the cross-linking degree induces

From the above, it is clear that FTIR spectroscopy is a sensitive and convenient tool to study the physicochemical modifications of bone composition regarding the mineral phase as well as the organic matrix. The detailed information provided by this technique is extremely

during demineralization (Horneman et al., 2004; Lewandrowski et al., 1996, 1997).

bands and by the similarity with the spectrum of collagen.

the resultant material, mostly composed of collagen.

higher retaining of the organic content in the composite.

**5. Conclusions** 

Fig. 12. Cumulative intrusion curve of human cortical bone, before (control) and after calcination at 600, 900 and 1200 ºC, measured by mercury porosimetry (Figueiredo et al., 2010).

demineralization procedures that naturally result in products with different properties (Bae et al., 2006; Eggert & Germain, 1979; Y. P. Lee et al., 2005; Lomas et al., 2001; Peterson et al., 2004). As reported in a recent study about BMPs depletion in particles of bovine cortical bone under acid exposure (0.25 and 0.5 M HCl) (Pietrzak et al., 2009), the availability of BMP-7 increases as demineralization occurs but, after reaching a maximum in the extraction bath, continuously declines. These results alert for the need to control the demineralization processing conditions. Normally, the process of bone demineralization is carried out by immersing the sample in a variety of strong and/or weak acids. In the case of using HCl (the most frequently used), the major inorganic constituent of bone (hydroxyapatite) reacts to form monocalcium phosphate and calcium chloride (Dorozhkin, 1997; Horneman et al., 2004).

FTIR has been used to monitor the bone demineralization process using HCl under different experimental conditions (acid concentrations and exposure times) (Figueiredo et al., 2011). Fig. 13 shows the FTIR spectra of bone samples (¼ of a ring of a human femoral diaphysis after being transversely cut into rings of approximately 1 cm width) submitted to demineralization with HCl 1.2 M for different periods of time. From the analysis of the FTIR spectra of the surface and of the core of the bone blocks, it was confirmed that the demineralization starts at the surface (absence of 1, 3 PO4 3- bands and relative increase of the

Fig. 13. FTIR spectra from the outer surface and from the core of the human bone samples after immersion in 1.2 M HCl for different time intervals. Demineralization proceeds from the surface into the core of the bone samples, as evidenced by the absence of phosphate bands and by the similarity with the spectrum of collagen.

collagen bands intensity) and progresses to the interior of the samples. These results support the concept of a diffusion model and agree with the proposed theory of the unreacted core during demineralization (Horneman et al., 2004; Lewandrowski et al., 1996, 1997).

This study was complemented with kinetic profiles and analysis of the samples´ structural modifications. As expected, increasing the acid concentration led to an increase in the demineralization rate, but not in a proportional way. In addition, microscopic observations demonstrated that despite the structural deformation resultant from demineralization, the basic bone microstructure was preserved. The loss of mineral led to a progressive reduction of mechanical strength and an increase of plastic properties (e.g. flexibility and elasticity) of the resultant material, mostly composed of collagen.

Although the deterioration of the organic component of bone was not examined in detail in this work, other studies using FTIR to analyse the effect of acids on the composition and structure of collagen during extraction from different tissues, may provide useful information on that subject. In fact, acid treatment of collagen samples was found to originate reduction of intermolecular cross-linking and hydrolysis of peptide bonds, as evidenced after curve-fitting in the spectral regions of the Amide I, II and III bands of collagen (Muyonga et al., 2004). These changes in collagen composition explained the observed loss of structural order. In addition, the amount and characteristics of the extracted fragments of collagen was related with the experimental conditions. These results agree with those from a FTIR study concerning the cross-linking of a collagen-hydroxyapatite nanocomposite with glutaraldehyde, as a model for the bone matrix (Chang & Tanaka, 2002). The spectral analysis showed that the increase of the cross-linking degree induces higher retaining of the organic content in the composite.

#### **5. Conclusions**

332 Infrared Spectroscopy – Life and Biomedical Sciences

Fig. 12. Cumulative intrusion curve of human cortical bone, before (control) and after calcination at 600, 900 and 1200 ºC, measured by mercury porosimetry (Figueiredo et al.,

demineralization procedures that naturally result in products with different properties (Bae et al., 2006; Eggert & Germain, 1979; Y. P. Lee et al., 2005; Lomas et al., 2001; Peterson et al., 2004). As reported in a recent study about BMPs depletion in particles of bovine cortical bone under acid exposure (0.25 and 0.5 M HCl) (Pietrzak et al., 2009), the availability of BMP-7 increases as demineralization occurs but, after reaching a maximum in the extraction bath, continuously declines. These results alert for the need to control the demineralization processing conditions. Normally, the process of bone demineralization is carried out by immersing the sample in a variety of strong and/or weak acids. In the case of using HCl (the most frequently used), the major inorganic constituent of bone (hydroxyapatite) reacts to form monocalcium phosphate and calcium chloride (Dorozhkin, 1997; Horneman et al.,

FTIR has been used to monitor the bone demineralization process using HCl under different experimental conditions (acid concentrations and exposure times) (Figueiredo et al., 2011). Fig. 13 shows the FTIR spectra of bone samples (¼ of a ring of a human femoral diaphysis after being transversely cut into rings of approximately 1 cm width) submitted to demineralization with HCl 1.2 M for different periods of time. From the analysis of the FTIR spectra of the surface and of the core of the bone blocks, it was confirmed that the

3- bands and relative increase of the

demineralization starts at the surface (absence of 1, 3 PO4

2010).

2004).

From the above, it is clear that FTIR spectroscopy is a sensitive and convenient tool to study the physicochemical modifications of bone composition regarding the mineral phase as well as the organic matrix. The detailed information provided by this technique is extremely

Characterization of Bone and Bone-Based Graft Materials Using FTIR Spectroscopy 335

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Additionally, FTIR has been extensively used to characterize natural or synthetic graft materials, as well as to monitor the properties of the new bone formation. Furthermore, since the composition and the morphostructural parameters of a bone graft affect their biocompatibility, biodegradation and ultimately their osteointegration, the use of FTIR spectroscopy (including FTIRM and FTIRI) allows an interdisciplinary approach between chemists, molecular biologists and medical investigators.

#### **6. References**


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**19** 

*Japan* 

*Nihon University* 

**Brain-Computer Interface Using** 

**Near-Infrared Spectroscopy for Rehabilitation** 

Currently, the Brain Computer Interface (BCI) is being studied vigorously. BCI extracts thoughts in the human brain as cranial nerve information and uses the information as inputs to control machinery and equipment. Fig. 1 describes schematic BCI system. If this system enables operating machinery and equipment directly from cranial nerve information without the subject moving his or her hands and feet, it can be applied to care-taking robots

BCI systems can be divided into two forms. The invasive form reads cranial nerve information using electrodes embedded directly into the brain. The non-invasive form reads cranial nerve activity from the surface of the head using near infrared spectroscopy (NIRS) or electroencephalography (EEG). as an example of invasive form, Donoghue LR. et al. extracted nerve activity of primary motor area and controlled a robot hand and mouse cursor (Hochberg LR et. Al, 2006). Though the invasive form has high signal accuracy, it

Recognition

Data

**Extraction/recognition**

imposes a heavy load on the user (e.g., surgery and infections after surgery).

and rehabilitation for physically handicapped individuals.

**Measurement**

Real-time signal processing

> Analysis Extraction

**1. Introduction** 

Fig. 1. Schematic of BCI system

Kazuki Yanagisawa, Hitoshi Tsunashima and Kaoru Sakatani

Results

Controller Equipment

**Control**

Control signal

