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Materials Science and Engineeri

The evaluation of collagen gel with various connection states by using MRI

Hiroki Kudo a,⁎, Naoki Mukai a, Chen Gouping b, Tomokazu Numanno c, Kazuhiro Honma c,Tetsuya Tateishi b, Yutaka Miyanaga b, Syumpei Miyakawa a

a Doctoral Program of Sports Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai,Tsukuba, Ibaraki, 305–8577, Japan

b National Institute for Materials Science, Ibaraki, Japanc National Institute of Advanced Industrial Science and Technology(AIST), AIST Tsukuba East, Ibaraki, Japan

Received 10 October 2005; accepted 12 October 2006Available online 24 January 2007

Abstract

To noninvasively evaluate the connection states of collagen fiber, a characterizing factor of the physical property, is considered to be helpful inthe evaluation of cartilage functions. The purpose of this study was to examine how the connection states of collagen influence the MRIparameters by evaluating the collagen gel with various connection states using MRI. MRI was performed to six type I collagen gel samples withvarious connection status and a water sample. The evaluation parameters included T1 relaxation time, T2 relaxation time, and diffusioncoefficient. With regard to gel samples with cross-links, the T2 relaxation time was shortened in proportion to the dose of glutaraldehyde. It isconsidered that as the glutaraldehyde concentration increases, the distance between protons in water molecules decreases; this is followed by astronger bipole–bipole interaction, resulting in a shorter T2 relaxation time. The diffusion coefficient for gel samples with cross-links alsodecreased with increasing glutaraldehyde concentrations. However, gel samples without glutaraldehyde were almost the same as that of the water.This result suggested that the degree of entrapment of water inside the gel samples without cross-links, even when it converted into gel, was foundto be nearly equal to that of the free water.© 2007 Published by Elsevier B.V.

Keywords: Magnetic Resonance Imaging; Collagen gel; cross-link; T1 relaxation time; T2 relaxation time; Diffusion coefficient

Table 1Composition of the samples

Sample 0.5%type‡Tcollagen(ml)

PBS(ml)

Glutaraldehyde(ml)

Water(ml)

Total(ml)

1) 12 1.5 1.5 0 152) 12 1.5 0.75 0.75 153) 12 1.5 0.15 1.35 154) 12 1.5 0.075 1.425 155) 12 1.5 0 1.5 15

1. Introduction

The major components of the extracellular matrix of anarticular cartilage are collagen fibers and proteoglycans. Insidecartilaginous tissues, a large amount of water is retained byhydrated proteoglycans located in the gaps of strong collagenfiber networks. The strong collagen networks provide kineticsupport to the cartilaginous tissues, and their entrapments ofproteoglycans containing a large amount of water establish thephysical and functional properties of articular cartilages, such asanti-extensibility and anti-compressibility. The collagen fibersmaintain their network structures by forming cross-links [1–3].It is known that when the collagen networks begin to collapsesuch as degeneration, proteoglycans increase their degree of

⁎ Corresponding author. Tel./fax: +81 29 853 5600(8362).E-mail address: kudo@med.taiiku.tsukuba.ac.jp (H. Kudo).

0928-4931/$ - see front matter © 2007 Published by Elsevier B.V.doi:10.1016/j.msec.2006.10.012

freedom and retained water content, causing a change in thecartilage viscoelasticity.

With regard to magnetic resonance imaging (MRI), differenttypes of imaging procedures have been developed until now;

6) 12 0.75 0 2.25 157) 0 0 0 20 20

1)–4): collagen samples with cross-links.5), 6): collagen samples without cross-links.7): Ultrapure water.

Fig. 1. Collagen gel samples and ultrapure water sample for MR imaging. Eachsample 1)–7) in the test tube with the screw entrance were grouped for MRimaging.

Fig. 2. T1 relaxation time for gel samples and ultrapure water. There are nosignificant difference between each gel samples and the ultrapure water.

271H. Kudo et al. / Materials Science and Engineering C 28 (2008) 270–273

these imaging procedures enable a more detailed morphologicalobservation of articular cartilages, showing the laminarstructures by T2 weighted images [4–7] and changes in theT1 relaxation time and diffusion coefficient due to the decreasein proteoglycans [8]. However, the evaluation of the connectionstates of collagen has not yet been reported. To noninvasivelyevaluate the connection states of collagen fiber, a characterizingfactor of the physical property is considered to be helpful in theevaluation of cartilage functions.

The purpose of this study was to evaluate collagen gel invarious connection states with MRI and to examine how theconnection states of collagen influence the MRI parameters.

2. Materials and methods

2.1. Materials

Three sets of seven gel and water samples 1)–7) were usedin this study (Table 1) (Fig. 1). Gel samples 1)–4) with cross-links were prepared using 0.5% of pepsin-soluble type Icollagen prepared from bovine skin, phosphate bufferedsaline (PBS), glutaraldehyde, and water. Gel samples 5) and6) without cross-links were prepared using collagen, PBS, andwater. The sample 7) comprised degassed ultrapure water. Gelsamples 1)–4) differed in the number of cross-links withfour increased glutaraldehyde contents, and gel samples 5) and

Table 2Results of T1, T2 relaxation time and diffusion coefficient

Sample T1 relaxation time s(mean/SD)

T2 relaxation times (mean/SD)

Diffusioncoefficient×10−3mm2/s

1) 2.30/0.22 0.91/0.02 a 1.90/0.052) 2.38/0.13 0.99/0.06 a 1.97/0.053) 2.39/0.19 1.37/0.31 a,⁎,⁎⁎ 2.06/0.07⁎,⁎⁎

4) 2.40/0.18 1.58/0.12 a,⁎,⁎⁎ 2.09/0.06⁎,⁎⁎

5) 2.40/0.22 1.57/0.10 a,⁎,⁎⁎ 2.12/0.04⁎,⁎⁎

6) 2.38/0.08 1.55/0.22 a,⁎,⁎⁎ 2.12/0.03⁎,⁎⁎

7) 2.43/0.18 2.05/0.11 2.12/0.03⁎,⁎⁎

⁎⁎Significantly different from gel sample 1) ( pb0.05).⁎⁎⁎Significantly different from gel sample 2) ( pb0.05).a Significantly different from ultrapure water 7) ( pb0.05).

6) were prepared by changing the PBS amount. The collagenconcentration in each gel sample remained constant at 0.4%.

2.2. MRI

MR imaging was performed with 2.0T superconductingmagnet (Biospec 20/30 system; Bruker Inc., Karlsruhe,Germany) and a 72-mm birdcage coil. ParaVision (BrukerInc., Karlsruhe, Germany) software was employed in this study.The evaluation parameters included T1 relaxation time, T2relaxation time, and diffusion coefficient. The saturation re-covery method was employed for calculating the T1 relaxationtime. Seventeen phases of repetition time (TR), namely, 100,150, 200, 250, 300, 350, 400, 500, 750, 1000, 1500, 2000, 3000,5000, 7500, 10,000, and 15,000 ms, were set along with an echotime (TE) of 15 ms and a matrix of 128×128. To measure the T2relaxation time, imaging was performed using multi-spin echosequence with a TR of 15,000 ms; thirty phases of TE, namely,15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210,225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405,420, 435, and 450 ms; and a matrix of 64×64. Conventionaldiffusion weighted spin echo sequence was performed forcalculating the diffusion coefficient. Since the water diffusionphenomenon in the samples were isotropic self-diffusion,

Fig. 3. T2 relaxation time for gel samples and ultrapure water. Significantdifference between gel samples 1) – 6) and 7) is indicated ( pb0.05).⁎Significantly different from sample 1) ( pb0.05). ⁎⁎Significantly differentfrom sample 2) ( pb0.05).

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motion proving gradient (MPG) were applied to three axes,namely, x, y, and z. The imaging parameters included a TR of10,000 ms, TE of 38.1 ms, MPG separation of 18.98 ms, anddiffusion gradient duration of 10.5 ms. Five phases of b valuerepresenting the intensity of the effect of MPG, namely, 0.537,72.009, 267.131, 585.904, and 1028.326 were used along withthe matrix of 128×128. The other imaging parameters, namelythe field of view and thickness were commonly used in eachsequence and were set at 70 mm and 3 mm, respectively. Theacquisition times required for saturation recovery for calculatingthe T1 relaxation time, multi-spin echo sequence for measuringthe T2 relaxation time measurement, and diffusion weightedspin echo sequence were 1 h 42 min 24 s, 32 min 02 s, and 1 h46 min 40 s, respectively. All sample experiments were per-formed at room temperature of 23 degree centigrade (Table 2).

Image J, an image analysis software, was employed for an-alyzing the obtained graphics in order to calculate the T1 relax-ation time, the T2 relaxation time, and the diffusion coefficient.

The one-way factorial ANOVA and the Fisher's PLSD testwere used in statistical comparisons. A p value of b0.05 waschosen to indicate significance.

3. Results

The T1 relaxation times for gel samples 1), 2), 3),4), 5), 6), and 7) were 2.30 +/−0.22 s, 2.38 +/−0.13 s,2.39 +/−0.19 s, 2.40 +/−0.18 s, 2.40 +/−0.22 s, 2.37 +/−0.08 s,and 2.43 +/−0.18 s, respectively; no significant difference wasobserved in the T1 relaxation time among all these samples(Fig. 2).

The T2 relaxation times for gel samples 1), 2), 3), 4), 5),6), and 7) were 0.91 +/−0.02 s, 0.99 +/−0.06 s, 1.37 +/−0.31 s,1.58 +/−0.12 s, 1.57 +/−0.10 s, 1.49 +/−0.31 s, and 2.05 +/−0.11 s, respectively. The T2 relaxation times for gel samples 1)–6)were significantly lower than that of the ultrapure water sample 7).With regard to gel samples with cross-links, the T2 relaxation timefor gel samples 1)–4) shortened in proportion to the glutaralde-hyde, a cross-link agent, concentration of the samples (Fig. 3).

The diffusion coefficients for samples 1), 2), 3), 4), 5), 6), and7) were 1.90 +/−0.05×10−3 mm2/s, 1.97 +/−0.05×10−3 mm2/

Fig. 4. Diffusion coefficient for gel samples and ultrapure water. ⁎Significantlydifferent from gel sample 1) ( pb0.05), ⁎⁎Significantly different from gelsample 2) ( pb0.05).

s, 2.06 +/−0.07×10−3 mm2/s, 2.09 +/−0.06×10−3 mm2/s,2.12 +/−0.04×10−3 mm2/s, 2.12 +/−0.03×10−3 mm2/s, and2.12 +/−0.03×10−3 mm2/s, respectively. The diffusion coeffi-cients for gel samples 1)–4) with cross-links decreased withincreasing glutaraldehyde concentrations, which was a similartendency to the case of the T2 relaxation time. However, unlikethe case of the T2 relaxation time, the diffusion coefficients forgel samples 5)–6) without cross-links were almost the same asthat of the ultrapure water (Fig. 4).

4. Discussion

4.1. T1 relaxation time

The proton density and the water status of biological tissueare regulating factor to provide for the T1 relaxation time.The proton density depends on the tissue water content in theorganization and it is shown that there is a positive correlationbetween the water content rate and the T1 relaxation time inthe cartilaginous tissue [9]. The T1 relaxation time of boundwater (hydration layer water) proton is shorter than bulkwater (free water) proton via the macromolecular hydrationeffect.

In this study, the result showed no significant difference inthe T1 relaxation time among the gel and water samples. Thisresult might be attributed to the low macromolecular hydrationeffect because of low concentration of additive collagen, PBS,and glutaraldehyde and the similar proton density due to thesame degree of water content rate among the samples.

4.2. T2 relaxation time

T2 relaxation time is influenced by water content of thetissue, T1 relaxation time, a structure of macromolecules,and water status (hydrate status of macromolecules). In therelationship between T2 relaxation time and structure ofmacromolecules, T2 relaxation time varies according to theangle between the direction of collagen fiber orientation andstatic magnetic field due to the dipole–dipole interaction[6,7,10]. When the water status between bound water and bulkwater is different, the correlation time of water moleculesmovement is also different. In bound water, the T2 relaxationtime is shortened by limitation of the water molecule move-ment due to macromolecules. However, according to the resultof the T1 relaxation time, there were no differences on thewater content and the T1 relaxation time among the samples.Moreover, the collagen fiber orientation of the gel samplesused in this study was not controlled. Therefore, the observedshortening of T2 relaxation time that depended on the glu-taraldehyde concentration of the gel samples was not caused bythe T1 relaxation time, water content, or the collagen fiberorientation. Instead, it reflected the difference in the degree ofentrapment of water. In other word, as the glutaraldehyde(cross-links) concentration increased, the distance betweenprotons in water molecules decreased, and the dipole–dipoleinteraction became stronger, which result in the shorter T2relaxation time.

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4.3. Diffusion coefficient

Diffusion is a physical phenomenon that is independentof MRI tissue parameters such as T1 or T2 relaxation time.Through the diffusion, we can obtain the MR signals reflectingmicrostructure such as tissue structure, physical properties ofeach tissue component, microstructures of the biological tissue,and stereostructures, whose images used to be difficult toobtain. The difference of the diffusion coefficient that decreasedaccording to the glutaraldehyde concentration among the gelsamples 1) – 4) with cross-links implies that the collagennetwork becomes stronger on the basis of the number of cross-links, which results in the stronger entrapment of water in thenetwork. In the early stage of osteoarthritis, the collagen net-work becomes loose and the proteoglycans inside the cartilagesgain a degree of freedom, which causes softening of the articularcartilages. It is said that this initial change is occurred due to thedegradation of type IX collagen, which plays an important roleas a cross-link agent in the maintenance of collagen networkstructures [11,12]. The obtained result that the diffusion co-efficient changed according to the number of cross-links sug-gests that in the early stage of osteoarthritis, the change in thediffusion coefficient might be related to the collapse and de-pletion of cross-links in addition to the decrease in proteogly-cans. It was intriguing that the diffusion coefficients of gelsamples 5)–6), which had only hydrogen bonds but not cross-links, was almost the same as that of the ultrapure water. Thedegree of restriction of water inside the gel samples 5)–6), evenwhen it converted into gel, was found to be nearly equal to thatof the free water. The result indicates that the diffusion co-efficient might be used as an indicator when the maturationprocess of cultured cartilage is estimated by using MRI.

5. Conclusion

We evaluated the collagen gel with andwithout cross-links byusing MRI. No significant difference was observed in the T1

relaxation time among all these samples. The T2 relaxation timesfor gel samples were significantly lower than that of the ultrapurewater sample. Furthermore, the T2 relaxation time for gel sam-ples with cross-links showed lower values along with theincrease of cross-links. The diffusion coefficient for gel sampleswith cross-links also decreased with increasing glutaraldehydeconcentrations. This result suggested that the diffusion coeffi-cient seems to be sensitive to the collapse and depletion of cross-links in addition to the decrease in proteoglycans in the earlystage of osteoarthritis.

In addition, gel samples without glutaraldehyde were almostthe same as that of the water. This result suggested that thedegree of entrapment of water inside the gel samples withoutcross-links, even when it converted into gel, was found to benearly equal to that of the free water. This result indicates thatthe diffusion coefficient might be used as an indicator when thematuration process of cultured cartilage is estimated by usingMRI.

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