2.1.

Preparation of Collagen Lattices

The test specimens were collagen lattices containing 0.3% (w/v) collagen supplemented with 20% w/v GAG. Lattices were formed by mixing $4.29\mathrm{mg}∕\mathrm{ml}$ type I collagen solution acid extracted from rat tail tendon,²¹ a mixture of $10\times \text{Dulbecco}$ ’s Modified Eagle’s Medium (DMEM) and $0.4\mathrm{M}$ NaOH (2:1), and the GAG, $\mathrm{C}\mathrm{H}6\mathrm{S}{\mathrm{O}}_4$ , prepared at $3\mathrm{mg}∕\mathrm{ml}$ in $1\times \text{serum}$ -free DMEM, at a ratio of 7:1:2. The pH was adjusted to 8 to 8.5 by dropwise addition of $1\mathrm{M}$ NaOH.

Five ml aliquots of the collagen lattice solution were pipetted into a polycarbonate (Stockline Plastics Ltd., Glasgow, UK) cell chamber measuring $24\times 60\times 11\mathrm{mm}$ with an outer diameter of $100\mathrm{mm}$ . In order to have a hydrophobic cell chamber to inhibit cell attachment, the cell chamber was siliconized for $24\mathrm{h}$ in a fume cupboard using Sigmacote. Before cell seeding, the collagen lattices were washed twice by incubating them in complete DMEM containing 10% v/v fetal calf serum, $50\text{units}∕\mathrm{ml}$ penicillin/ $50\mu \mathrm{g}∕\mathrm{ml}$ streptomycin, and nonessential amino acids. 3T3 mouse fibroblasts were seeded onto the set collagen lattice at a density of ${10}^4\text{cells}∕{\mathrm{cm}}^2$ in $7\mathrm{ml}$ of complete DMEM and incubated for $4\mathrm{h}$ at $37°\mathrm{C}$ (Heraeus Electronics, Hanau, Germany) in an atmosphere of 5% $\mathrm{C}{\mathrm{O}}_2$ in air to allow cell attachment. After this time period, the FPCLs were detached from the walls of the cell chamber and floated freely in the culture medium. Laser treatment was carried out after this $4\text{-}\mathrm{h}$ attachment period. Thereafter, cells were cultured for up to $7\text{days}$ with a daily change of $2\mathrm{ml}$ of the $7\mathrm{ml}$ culture medium. Collagen lattices were also cultured in the absence of cells and laser treated by following the same protocol as described earlier, but omitting the cells.

2.2.

Laser Irradiation

Lattices were irradiated using a $5\text{-}\mathrm{mW}$ continuous wave, linearly polarized He-Ne laser (3225H-PC, Hughes Aircraft Co.) operating at $632.8\mathrm{nm}$ with a beam profile at TEM00 mode delivering a laser beam with a spot size of $1.5\mathrm{mm}$ . The output power was measured using Molectron MAX 5200 and Metrologic power meters calibrated at $632.8\mathrm{nm}$ . The body of the laser was attached to a scanner, which was controlled by two linear actuator stepper motors. A host personal computer with a Pentium II processor running on LabVIEW 6.0 (Laboratory Virtual Instrument Engineering Workbench, National Instruments Corp.) and a development environment based on the graphical programming language G were used to control the scanning speed of both motors, $V$ (cm/s), which determined the laser fluence or energy density, $\rho$ $(\mathrm{J}∕{\mathrm{cm}}^2)$ , the relation being:

The laser, delivering an output power of $3.5\mathrm{mW}$ , was positioned $20\mathrm{mm}$ above the surface of the specimen. A holder specially fabricated to accommodate the cell chamber was attached to the top of the angular table together with a linkage connecting the linear micrometer with the $x\text{-}y$ table, and an alignment rail enabled precise positioning of the fibroblast-populated collagen lattices (FPCL) for LLLT treatment (Fig. 1 ). The entire computer-assisted experimental setup was housed in a controlled environment of $37°\mathrm{C}$ .

Fig. 1

The laser scanning system.

2.3.

Analysis of Cell Viability, Morphology, Mitochondrial Membrane Depolarization, and Collagen Fiber Content of the Matrix

To examine the viability of the fibroblasts in the FPCL, ethidium bromide (EB) and 5-carboxyfluorescein diacetate (CFDA) staining was used.²² CFDA stains the viable 3T3 fibroblasts green, and EB stains the nuclei of cells with damaged membranes red. The lattices were washed twice with phosphate buffered saline (PBS), and then $2\mathrm{ml}$ of 0.1% (w/v) EB in PBS was added. The lattices were kept in the dark at room temperature for $6\min$ before washing them again three times with PBS. Three ml of $25\mu \mathrm{M}$ CFDA in PBS was then added, and the lattices were kept in the dark at $4°\mathrm{C}$ . After $15\min$ , the lattices were again washed three times with PBS and viewed under the confocal laser scanning microscope (Leica CLSM). For the detection of cytoskeletal damage, fluorescein isothiocyanate (FITC)-labeled phalloidin was used to stain the actin, as described by Wulf ²³ The lattices were fixed by immersing them in PBS containing 4% formaldehyde for $20\min$ at room temperature. Fixed samples were then washed three times with PBS and incubated with 1:500 dilution of FITC-phalloidin for $1\mathrm{h}$ in the dark at room temperature, washed again three times with PBS, and examined by CLSM. To visualize the collagen fibers in the collagen-GAG lattices, both the LLLT-treated and control lattices were immersed for $5\min$ at room temperature in 0.1% (w/v) acriflavine, a mixture of 3,6-diamino-10-methylacridinium (acriflavine) and 3,6-diaminoacridine (proflavine). Samples were washed three times with PBS to remove excess stain before viewing on the CLSM. Examination by CLSM for CFDA/EB, FITC-phalloidin, and acriflavine stained samples used $488\text{-}\mathrm{nm}$ excitation with a $510\text{-}\mathrm{nm}$ dichroic beamsplitter in place. Data from the two channels were collected, using a barrier filter of $590\mathrm{nm}$ in channel 1 and a bandpass filter of $530\mathrm{nm}$ in channel 2. The objective lens was a $25\times \text{water}$ immersion lens (NA 0.75).

To determine the mitochondrial membrane potential, accumulation of the fluorescent potentiometric dye tetramethylrhodamine ethyl ester (TMRE) was measured in the control and LLLT-treated 3T3 cells. For this method, the cells were cultured on polystyrene $60\text{-}\mathrm{mm}$ Petri dishes and not in collagen lattices. Cells were seeded at ${10}^4\text{cells}∕{\mathrm{cm}}^2$ in $7\mathrm{ml}$ complete DMEM and treated with the laser after allowing $4\mathrm{h}$ for attachment as with the FPCL samples. As a positive control, cultured cells were also incubated with $100\mu \mathrm{M}$ carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) for $1\mathrm{h}$ at $37°\mathrm{C}$ to disrupt the mitochrondrial membrane potential. Cultures of control, LLLT-treated, and FCCP-treated cells were incubated with $100\mathrm{nM}$ TMRE in complete DMEM for $20\min$ , after which they were washed three times with PBS and imaged by $z$ -sectioning. Each section was $2\mu \mathrm{m}$ , and a total of 18 sections were imaged. The images were collected using Lasersharp 2000 software (Bio-Rad Laboratories) running OS/2. Images were recorded using an MRC 1024 CLSM (Bio-Rad) coupled to a Nikon E600FN upright microscope using a $60\times \mathrm{FLUOR}$ water dipping lens $(\mathrm{NA}=10)$ . TMRE was excited at $568\mathrm{nm}$ using a Krypton/Argon laser. A $580\text{-}\mathrm{nm}$ LP filter (Chrom, Inc.) was used to collect emission of light greater than $580\mathrm{nm}$ . Time-dependent TMRE fluorescent changes were measured in frame scan mode. Projections of the image sections were created using a “maximum” alogarithm in the Bio-Rad Lasersharp 2000 software. Subsequently, the intensity of the cells was quantified in Scion Image.

2.4.

Measurement of Contraction

The area of the lattices in all the experiments was measured daily using a light box over which the cell chambers were placed on a standard millimeter grid. The area of the specimen was then calculated using digital planimetry and imaging software (Scion Image, www.scioncorp.com).

Using Scion Image, the file containing the digitized image of the collagen lattices was opened, the perimeter of the cell chamber outlined, and the original area calculated. The areas of the contracted lattices were measured in a similar way by first plotting the outline of the contracted lattices using a freehand selection tool in Scion Image and then instructing the program to measure the outlined area. The contracted area of the lattices was expressed as a percentage of the original area.

3.1.

Lattice Contraction

Both the unseeded collagen lattices and the fibroblast populated lattices contracted, but the magnitude and time scale of the contraction were markedly different.

Seeded lattices showed progressive contraction over the $7\text{days}$ for which it was monitored (Figs. 2 and 3 ). The rate of contraction was greatest initially, during the first day of monitoring, and then it decreased nonlinearly. The maximum contraction occurred on day 7, the last day of monitoring, and this value was used to characterize the contraction of the FPCL. Collagen lattices in the absence of cells reached a maximum contraction on the second day and remained constant thereafter (Figs. 2 and 3).

Fig. 2

Contraction of control and LLLT-treated unseeded collagen lattices and FPCL at $2\mathrm{J}∕{\mathrm{cm}}^2$ . Results are expressed as percentages of the original area of the collagen lattices and are the mean $\text{values}\pm \text{standard}$ deviation (SD) of five repeated experiments. Diamonds indicate control (FPCL); triangles indicate control (collagen lattices); squares indicate LLLT at $2\mathrm{J}∕{\mathrm{cm}}^2$ (FPCL); and circles indicate LLLT at $2\mathrm{J}∕{\mathrm{cm}}^2$ (collagen lattices).

Fig. 3

Contraction of control and LLLT-treated unseeded collagen lattices and FPCL at $3\mathrm{J}∕{\mathrm{cm}}^2$ . Results are expressed as percentages of the original area of the collagen lattices and are the $\text{mean}\pm \mathrm{SD}$ , $n=5$ . ${}^{*}p<0.05$ , comparing values from the LLLT-treated lattices with and without cells with those from untreated seeded and unseeded collagen lattices, respectively, by two-tailed Student’s $t$ -test. Diamonds indicate control (FPCL); triangles indicate control (collagen lattices); squares indicate LLLT at $3\mathrm{J}∕{\mathrm{cm}}^2$ (FPCL); and circles indicate LLLT at $3\mathrm{J}∕{\mathrm{cm}}^2$ (collagen lattices).

The values of the parameters used to characterize the contraction of the lattices in control and LLLT-treated samples are shown in Table 1 . Comparison of the values for control and laser-treated specimens using two-tailed Student’s $t$ -test showed that irradiation at fluences of 1, 2, and $4\mathrm{J}∕{\mathrm{cm}}^2$ had no statistically significant effect. The effect of $2\mathrm{J}∕{\mathrm{cm}}^2$ is shown on Fig. 3. Irradiation at $3\mathrm{J}∕{\mathrm{cm}}^2$ produced significantly greater contraction of the unseeded lattices and reduced contraction of FPCL compared with values from the control unseeded collagen lattices and FPCL, respectively $(p<0.05)$ . The increase in contraction of the unseeded lattices was significant for all days after day 1, but the decrease in contraction of FPCL was significant for only the last $3\text{days}$ of the monitoring period (Fig. 3).

Table 1

Contraction of FPCL and unseeded collagen lattices. Results are expressed as a percentage of the original area of the collagen lattice and represent the values after 7days in culture. Values are means of five experiments, with the standard deviation in parentheses.  * Denotes that the differences between laser-treated and control values are statistically significant, by two-tailed Student’s t -test.

Using CLSM, no significant visual difference was observed in the morphology and viability of the cells using CFDA and EB between the control and LLLT-treated FPCL [Figs. 4a and 4b ]. Elongated fibroblastic morphology was maintained for up to $7\text{days}$ after treatment, with typical alignment of cells in one direction. There was no discernable alteration in the number of nonviable cells after LLLT treatment.

Fig. 4

CLSM images of the control FPCL (a) and the LLLT-treated FPCL (b) at $3\mathrm{J}∕{\mathrm{cm}}^2$ , on day 7.

3.2.

Actin Filaments

Figure 5 shows the staining with FITC-labeled phalloidin after treatment with 2 and $3\mathrm{J}∕{\mathrm{cm}}^2$ . Intracellular actin filaments are less pronounced in cells treated with $3\mathrm{J}∕{\mathrm{cm}}^2$ than in either the controls or the cells treated with $2\mathrm{J}∕{\mathrm{cm}}^2$ . There are few prominent fibers crossing the interior of the cells, and actin staining appears to be mainly around the cell perimeter. Figure 6 shows that LLLT treatment has no significant effect on the accumulation of TMRE within cells, indicating that it does not alter the mitochondrial membrane potential. In contrast, FCCP treatment markedly decreased TMRE accumulation.

Fig. 5

CLSM images of actin stained with FITC-phalloidin in control and LLLT-treated FPCL. (a) and (c) are control untreated FPCL; (b) and (d) were treated with 2 and $3\mathrm{J}∕{\mathrm{cm}}^2$ , respectively.

Fig. 6

TMRE fluorescence intensity of control and LLLT-treated $(3\mathrm{J}∕{\mathrm{cm}}^2)$ 3T3 fibroblasts cultured on Petri dishes. Results are means ± SD, $n=3$ experiments. ${}^{*}p<0.05$ , comparing FCCP-treated cells with controls, by two-tailed Student’s $t$ -test.

3.3.

Collagen Fibers

The effect of LLLT treatment on the collagen fibers was investigated in cell-free matrices in order to distinguish the effect of laser irradiation from cell-induced changes. CLSM images of acriflavine stained collagen fibers at a depth of $20\mu \mathrm{m}$ into the collagen-GAG matrices are shown in fig. 7 . The images show that there are fewer collagen fibers in the matrices treated with $3\mathrm{J}∕{\mathrm{cm}}^2$ than in either controls or in samples treated with $2\mathrm{J}∕{\mathrm{cm}}^2$ . This was observed in three separate experiments, and Fig. 8 illustrates the mean fluorescent intensities measured in all experiments. After treatment with $3\mathrm{J}∕{\mathrm{cm}}^2$ , there was a significant decrease in fluorescent intensities measured at a depth of $20\mu \mathrm{m}$ in the matrices.

Fig. 7

Distribution of collagen fibers at a depth of $20\mu \mathrm{m}$ in control [(a) and (c)] and LLLT-treated [(b) is $2\mathrm{J}∕{\mathrm{cm}}^2$ and (d) is $3\mathrm{J}∕{\mathrm{cm}}^2$ ] collagen-GAG lattices.

Fig. 8

Acriflavine fluorescent intensity of control and LLLT-treated collagen—GAG lattices at a depth of $20\mu \mathrm{m}$ using laser fluences of 2 and $3\mathrm{J}∕{\mathrm{cm}}^2$ . Results are $\text{mean}\pm \mathrm{SD}$ , $n=3$ . ${}^{*}p<0.05$ , comparing differences between control and LLLT-treated lattices using two-tailed Student’s $t$ -test.