**3. Effect of extracellular osmolality on glycosaminoglycan production and cell metabolism with time in culture**

The effect of change in extracellular osmotic pressure on the amount of GAG production has not been well understood. It is important to initially know what culture condition provides the optimal environment for generation of articular cartilage. Therefore, we conducted an in vitro study to investigate the effects of change in extracellular osmotic pressure on cartilage cell morphology, GAG production, and cartilage cell metabolism, using the alginate beads system for three-dimensional culture of articular chondrocytes (Negoro, et al., 2008). Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2, Medium osmolality was altered by NaCl addition over the range 270-570 mOsm and monitored using a freezing point osmometer.

**Figure 1.** Changes of nutrient supply rote and extracellular environment of cartilaginous tissue with aging. (A) Normal cartilaginous tissue, (B) Osteoarthritis (OA).(A) This schema shows a representation of healthy articular cartilage over‐ lying the subchondral trabecular bone. Although chondrocytes occupy less than 1% of articular cartilage, they are re‐ sponsible for maintaining the integrity of the extracellular matrix by balancing macromolecular synthesis with breakdown. The matrix that surrounds them confers a mechanically resilient surface to the articulating bones within joints, and comprises collagens (principally collagen II), other noncollagenous proteins, and proteoglycans. In addition to structural support and absorption of shock offered by the subchondral bone, its small vessels and probably the in‐ terstitial bone fluid in osteocyte canaliculi, provide important nutrition to the cartilage. (B) The right schema shows some cartilage erosion, as seen in OA. The number of chondrocytes decreases with aging, but it is unknown whether this decline is caused by apoptosis, or insufficient supply of nutrients from the end plate. Ossification of the subchon‐ dral bone (tide mark) occurs with aging and is one of the major causes of cartilaginous degeneration. This leads to deterioration of the extracellular environment in the cartilaginous tissue and causes cellular impairment that is fol‐ lowed by a decline of matrix metabolism, resulting in progression to osteoarthritis. The nutrient supply for cells and the extracellular environment of the cartilaginous tissue have a considerable influence on the outcome of treating os‐ teoarthritis by bioengineering techniques.

#### **3.1. Cell viability**

levels, with levels of oxygen or pH falling with increases in rates of cell metabolism or cell density. For the chondrocytes to remain viable, the levels of extracellular nutrients and pH must remain above critical values. Because disc cells obtain ATP primarily by glycolysis, glucose is a critical nutrient. The cells start to die within twenty-four hours if glucose concen‐ tration falls below 0.2 mM and the efficiency of glucose transport into the cell is likely reduced at this glucose concentration (Windhaber et al., 2003). The rate of cell death increases when pH levels are acidic. The cell viability is reduced even with adequate glucose at pH 6.0. The osmotic environment of chondrocytes in the articular cartilage changes with loading and pathologic states. The osmolality of the extracellular matrix is regulated by negatively charging the GAG chains of PGs which adjust ionic composition. Particularly, extracellular osmolality is control‐ led by negatively charged PGs. It is now evident that an increase in the concentration of PGs which control ionic composition causes an increase in the osmolality, and conversely, a decrease in PGs reduces osmolality (Maroudas, 1981). Maroundas et al. (1975) investigated the osmotic pressures in articular sections extending to the sagittal sections and reported that the osmotic pressure in the articular cartilage is about 370-400 mOsm and were decreased in the degenerated articular cartilage. Thus, it may be said that osmotic pressure gradient disturbance associated with reduced PGs is an important factor contributing to the development of disc degeneration. The results also suggest that standard culture mediums do not provide an

The physico-chemical environment created and maintained by chondrocytes in turn has a powerful effect on cartilaginous metabolism. However, the supply of nutrients from vascular systems at the subchondral bone to the cartilaginous tissue of osteoarthritis is likely to be affected, causing the extracellular environment to deteriorate and some cells in degenerate cartilage are senescent (Kühn, 2004). This environment is often neglected by it can strongly influence matrix turnover or the responses of chondrocytes to growth factors or other external stimuli. Such limitations apply to all avascular tissues including tissue engineered constructs.

**3. Effect of extracellular osmolality on glycosaminoglycan production and**

The effect of change in extracellular osmotic pressure on the amount of GAG production has not been well understood. It is important to initially know what culture condition provides the optimal environment for generation of articular cartilage. Therefore, we conducted an in vitro study to investigate the effects of change in extracellular osmotic pressure on cartilage cell morphology, GAG production, and cartilage cell metabolism, using the alginate beads system for three-dimensional culture of articular chondrocytes (Negoro, et al., 2008). Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2, Medium osmolality was altered by NaCl addition over the range 270-570 mOsm and monitored using

appropriate ionic and osmotic environment for chondrocytes.

**cell metabolism with time in culture**

546 Regenerative Medicine and Tissue Engineering

a freezing point osmometer.

After 2, 4 and 6 days of culture, the chondrocyte viability rate was 90% or higher in all of the 4 osmolality groups (Fig.2). So, chondrocyte viability was not modified by the difference in extracellular osmolality. However, confocal microscopy showed that the cells were the largest under 270 mOsm and became smaller with increasing osmotic pressure (Fig.3). Under transmission electron micrographs of chondrocytes, at 370 and 470 mOsm all cells appeared viable, with large nuclei, dotted with chromatin and abundant rough endoplasmic reticulum (Fig.4B, C). The cells appeared active throughout the beads. In the beads cultured at 270mOsm, however, all cells were swelling with numerous cytoplasmic vacuoles and lipid droplets (Fig. 4A). Many cells had blebbing and these cells undergoing oncosis were seen. Under 570mOsm, many cells were reduced in size and blebbing was visible in the nuclei (Fig 4D,5A,5B).

#### **3.2. GAG production and cell metabolism**

Osmotic environment of cells in cartilage tissues is altered significantly by loading and morbid conditions. The cartilage tissues sustain static load and prolonged cyclical loading all the time

**Figure 2.** The cell viability by manual counting using a Live/Dead assay kit containing fluorescent probes. After 6 days of culture, the survival rate of cells was 90.3 ± 8.7, 93.3 ± 11.5, 94.4 ± 9.6, and 93.3 ± 11.5% (mean ± SEM) respectively in the 270, 370, 470, and 570 mOsm groups. The percentage of live and dead cells in sections was similar for the high and low osmolality cultures.

and osmolality imbalance occurs in the articular cartilage. To overcome osmotic imbalance and acquire new equilibrium, fluid is exuded from the tissue, and the PG level, cation level, and osmotic pressure are increased as a result. The chondrocytes always sustains high osmotic pressure. When loading is removed the tissue, fluid is slowly absorbed in turn, and the normal osmotic status is recovered. Urban, et al. (1993) incubated chondrocytes isolated from the articular cartilage in commercially- available DMEM solutions set at 250-270 mOsm of osmolality for 2 hours. Their experiment showed that the chondrocytes swelled by about 30-40% in the above osmolality condition and chondrocytes incubated in a medium set at 350-400 mOsm for osmolarity were most close to the size of chondrocytes in the intact tissues and synthesized the highest amount of PG. Hopewell & Urban (2003) investigated the effect of extracellular osmolality on chondrocytes cultured in alginate beads. Their study showed decreased sulphate incorporation rate for the cells incubated at high osmolality for 4 hours, recovery of sulphate incorporation rate for the cells incubated at high osmolality for 24 and 48 hours, and a higher sulphate incorporation rate than the original level for the cells incubated

Importance of Extracellular Environment for Regenerative Medicine and Tissue Engineering of Cartilagious Tissue http://dx.doi.org/10.5772/55566 549

**Figure 3.** Confocal microscopy showed that the chondrocytes were greatest in the 270 mOsm group and diminished gradually along with the increases in osmolality. The diameters of cells measured obviously decreased in the higher osmolality groups (P<0.05). After 6 days of culture, the chondrocyte diameter was 9.9 ± 0.9, 8.4 ± 0.6, 7.4 ± 0.9, and 6.3 ± 0.8 µm (mean ± SEM) respectively in the 270, 370, 470, and 570 mOsm groups. The cell diameter was already established by 2 days of culture. The cell diameter increase with time in culture (P<0.05). (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolality on glycosaminoglycan production and cell metabolism of artic‐ ular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26: 527-533,2008.)

further. Based on these results, they indicated that chondrocytes are sensitive to osmolality and are able to adjust for high osmolality during short time. Bush, et al (2005). reported a single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolality at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress. And also, Erickson, et al (2001). indicated that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in intracellular calcium ion. Palmer, et al (2001). measured the aggrecan promoter activity and mRNA levels using bovine monolayer chondrocytes subjected to hyperosmotic loading for different time periods from 1 minute to 24 hours. They concluded the hyper-osmotic loading regulates aggrecan gene expression and cell size in isolated. Thus, mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways.

and osmolality imbalance occurs in the articular cartilage. To overcome osmotic imbalance and acquire new equilibrium, fluid is exuded from the tissue, and the PG level, cation level, and osmotic pressure are increased as a result. The chondrocytes always sustains high osmotic pressure. When loading is removed the tissue, fluid is slowly absorbed in turn, and the normal osmotic status is recovered. Urban, et al. (1993) incubated chondrocytes isolated from the articular cartilage in commercially- available DMEM solutions set at 250-270 mOsm of osmolality for 2 hours. Their experiment showed that the chondrocytes swelled by about 30-40% in the above osmolality condition and chondrocytes incubated in a medium set at 350-400 mOsm for osmolarity were most close to the size of chondrocytes in the intact tissues and synthesized the highest amount of PG. Hopewell & Urban (2003) investigated the effect of extracellular osmolality on chondrocytes cultured in alginate beads. Their study showed decreased sulphate incorporation rate for the cells incubated at high osmolality for 4 hours, recovery of sulphate incorporation rate for the cells incubated at high osmolality for 24 and 48 hours, and a higher sulphate incorporation rate than the original level for the cells incubated

and low osmolality cultures.

548 Regenerative Medicine and Tissue Engineering

**Figure 2.** The cell viability by manual counting using a Live/Dead assay kit containing fluorescent probes. After 6 days of culture, the survival rate of cells was 90.3 ± 8.7, 93.3 ± 11.5, 94.4 ± 9.6, and 93.3 ± 11.5% (mean ± SEM) respectively in the 270, 370, 470, and 570 mOsm groups. The percentage of live and dead cells in sections was similar for the high

**Figure 4.** Electron micrograms of nucleus pulposus cells in the centre of the beads.A. under 270mOsm, B. under 370mOsm, C. under 470mOsm, D. under 570mOsm.At 270mOsm, the cell was swelling with numerous vacuoles and cytoplasmic organelles destroyed were visible. This cell undergoing oncosis were seen. At 370 and 470mOsm, all cells appeared viable. At 570mOsm, the cell and nuclei was reduced in size and chromatin condensation was visible in the nuclei. (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolality on glycosaminoglycan pro‐ duction and cell metabolism of articular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26: 527-533,2008.)

In our study, GAG accumulation was measured using a modified dimethylmethylene blue (DMB) assay. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolality and hyper-osmolality, and cell deaths were often observed on electron microscopy. While total GAG in beads/ml of beads volume increased with the duration of culture, it was greatest in the 370 mOsm group and lowest in the 270 mOsm group during culture (Fig.6A). The total GAG in beads/ml of beads volume was greatest in the 370 mOsm group, being 0.298±0.035 (mean ± SEM) mg/ml at day 6. It was lowest in the 270 mOsm group than in the other osmolality groups, being 0.122±0.019 mg/ml. In the high osmolality group at 570 mOsm, the total GAG in beads/ml of beads volume was greater than that in the 370 and 470 mOsm group after 2 days of culture, while the percentage of increased diminished subsequently until day 6. Similarly the rate of GAG in beads per live cell was the highest in the 370 mOsm group during culture (Fig.6B). While the total GAG in beads/million cells increased with the duration of culture, it was greatest in the 370 mOsm group and lowest in the 270 mOsm group during up to 6 days of culture. In the high osmolarity group at 570 mOsm, the total GAG in beads/million cells was Importance of Extracellular Environment for Regenerative Medicine and Tissue Engineering of Cartilagious Tissue http://dx.doi.org/10.5772/55566 551

**Figure 5.** Under 570mOsm, Some cells undergoing apoptosis were seen. Cells with condensed and fragmented nuclei and condensed chromatin (apoptotic bodies) and with cytoplasmic organelles destroyed were visible. (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolarity on glycosaminoglycan production and cell me‐ tabolism of articular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26: 527-533, 2008).

greater than that in the 270 mOsm group after 2 days of culture, while the percentage of increased diminished subsequently until day 6. The cell cultured at 370 and 470 mOsm were thus more active and accumulated significantly more GAG than cells cultured at 270 and 570 mOsm with time. In this study, the cells incubated at 370 mOsm produced the greater amount of GAG, and the cells incubated at high osmolality for 2 days showed a similar trend for GAG production to the results of Hopewell's experiment (2003) using isolated articular cartilage. However, the cells incubated further for 6 days produced a lower amount of GAG in the condition of high osmolarity and showed the profile of cell death (apoptosis) under electron microscope. Thus, this study indicates that chondrocytes is unable to adjust for such nonphysiological conditions lasting for a long time and this phenomenon plays a critical role in the development of cartilage degeneration and resultant OA.

In our study, GAG accumulation was measured using a modified dimethylmethylene blue (DMB) assay. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolality and hyper-osmolality, and cell deaths were often observed on electron microscopy. While total GAG in beads/ml of beads volume increased with the duration of culture, it was greatest in the 370 mOsm group and lowest in the 270 mOsm group during culture (Fig.6A). The total GAG in beads/ml of beads volume was greatest in the 370 mOsm group, being 0.298±0.035 (mean ± SEM) mg/ml at day 6. It was lowest in the 270 mOsm group than in the other osmolality groups, being 0.122±0.019 mg/ml. In the high osmolality group at 570 mOsm, the total GAG in beads/ml of beads volume was greater than that in the 370 and 470 mOsm group after 2 days of culture, while the percentage of increased diminished subsequently until day 6. Similarly the rate of GAG in beads per live cell was the highest in the 370 mOsm group during culture (Fig.6B). While the total GAG in beads/million cells increased with the duration of culture, it was greatest in the 370 mOsm group and lowest in the 270 mOsm group during up to 6 days of culture. In the high osmolarity group at 570 mOsm, the total GAG in beads/million cells was

527-533,2008.)

550 Regenerative Medicine and Tissue Engineering

**Figure 4.** Electron micrograms of nucleus pulposus cells in the centre of the beads.A. under 270mOsm, B. under 370mOsm, C. under 470mOsm, D. under 570mOsm.At 270mOsm, the cell was swelling with numerous vacuoles and cytoplasmic organelles destroyed were visible. This cell undergoing oncosis were seen. At 370 and 470mOsm, all cells appeared viable. At 570mOsm, the cell and nuclei was reduced in size and chromatin condensation was visible in the nuclei. (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolality on glycosaminoglycan pro‐ duction and cell metabolism of articular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26:

> The lactate production was measured enzymatically and the rate of sulphate GAG synthesis was measured using a standard 35S-sulphate radioactive method. Fig.7A shows the effect of extracellular osmolalities on lactate production by chondrocytes, a marker for total energy production. The rate of lactate production per live cell significantly decreased with time in culture. Lactate production was significantly decreased in hypo-osmolality (270 mOsm) group compared with the other groups. Thus, cell metabolism was decreased with the duration of culture, but metabolic hypofunction persisted under hypo-osmolality. Similarly the rate of sulphate incorporation per live cell was the highest in the 370 mOsm group during culture, and was decreased with an increase in extracellular osmolality (Fig. 7B). It was the lowest in the hypo-osmolality (270 mOsm) group during culture. The cells cultured at 370 and 470 mOsm were more active significantly more sulphate incorporation per live cell than cells cultured at 270 and 570 mOsm. The rate of sulphate incorporation fell more steeply than lactate rates with time in culture.

**Figure 6.** Effect of extracelluler osmotic change on GAG accumulation/tissue volume (A) and GAG produced per mil‐ lion cells (B). This figure gives pooled data for 4 representative osmolality from 3 separate experiments for cells cul‐ tured by articular chondrocytes. (A) GAG accumulation/tissue volume was significantly increased at 370 and 470 mOsm with time in culture. This was the highest in the 370 mOsm group and the lowest in the hypo-osmolality group (270 mOsm) decreased after 6 days of culture. In the hyper-osmolality group (570 mOsm), the rate of GAG accumula‐ tion/tissue volume wasn't decreased after 2 days of culture when compared with the 370 mOsm group. Values are mean ± standard error. (Scheffe, \*: P<0.05). (B) GAG produced per million cells was significantly increased at 370 and 470 mOsm with time in culture (\*,#: P<0.05, Scheffe between between 2 and 6 days)] This was the highest in the 370 mOsm group and the lowest in the hypo-osmolality group (270 mOsm) decreased after 6 days of culture. In the hyperosmolality group (470 and 570 mOsm), the rate of total GAG produced per million cells wasn't decreased after 2 days of culture when compared with the 370 mOsm group. (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26: 527-533,2008).

In this study, the chondrocytes produced the highest amount of GAG in the osmolality condition of 370 mOsm after 2 and 6 days of culture. The amount of GAG production was obviously lower in the low osmolality cultures than in the culture at the optimal osmolarity close to that in the normal cartilage. On electron microscopy of chondrocytes cultured under varying levels of osmolality, cells under 370 mOsm generally showed normal nuclei and cytoplasm, while cells under hypo-osmolality presented oncotic changes, with cellular swelling and destructed organelles of the cytoplasm. On the other hand, cells cultured under hyper-osmolality were reduced in size and some cells underwent apoptosis. Manjo & Joris (1995) reported oncosis is a form of cell death accompanied by cellular swelling, organelle swelling, blebbing, and increase membrane permeability. They also showed that necrosis can occur after both forms (oncosis and apoptosis) of cell death. Therefore, its mechanism is based on failure of the ionic pumps of the plasma membrane induced by the changes of extracellular osmotic environment. Thus, our physiological and morphological study showed the articular chondrocytes is unable to adjust for such non-physiological conditions lasting for a long time and this phenomenon plays a critical role in the development of cartilage degeneration and resultant OA.

Importance of Extracellular Environment for Regenerative Medicine and Tissue Engineering of Cartilagious Tissue http://dx.doi.org/10.5772/55566 553

**Figure 7.** Effect of extracellular osmotic change on lactate production rate (A) and 35S-sulphate incorporation rate (B). (A) The rate of lactate production per live cell decreased with time in culture (+: P<0.05, 2 way ANOVA with repeated measures among 2,4 and 6 days). After 4 and 6 days of culture, lactate production was clearly decreased under hypoosmolality (270 mOsm) compared with other levels of osmolality. (Scheffe between 270 and 370 mOsm [#: P=0.405, ##,###: P<0.05], 370 and 470 mOsm [\*: P=0.707, \*\*: P=0.988, \*\*\*: P=0.997], or 370 and 570 mOsm [\$: P=0.738, \$\$: P=0.559, \$\$\$: P=0.083]). (B) Sulphate incorporation rates fall with time in culture (+: P<0.05, 2 way ANOVA with re‐ peated measures among 2,4 and 6 days). Values are mean ± standard error. It was clearly decreased in the hypo-os‐ molality (270 mOsm) groups compared with the 370 mOsm groups during culture. After 6 days of culture, lactate production was clearly decreased under the 570 mOsm groups compared with the 370 mOsm groups. (Scheffe be‐ tween 270 and 370 mOsm [#,##,###: P<0.05], 370 and 470 mOsm [\*: P=0.537,\*\*:P=0.446,\*\*\*:P=0.109], 370 and 570 mOsm [\$: P=0.98, \$\$:P=0.366, \$\$\$:P<0.05]). (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three dimensional culture system. Clin Exp Rheumatol 26: 527-533,2008).

**Figure 6.** Effect of extracelluler osmotic change on GAG accumulation/tissue volume (A) and GAG produced per mil‐ lion cells (B). This figure gives pooled data for 4 representative osmolality from 3 separate experiments for cells cul‐ tured by articular chondrocytes. (A) GAG accumulation/tissue volume was significantly increased at 370 and 470 mOsm with time in culture. This was the highest in the 370 mOsm group and the lowest in the hypo-osmolality group (270 mOsm) decreased after 6 days of culture. In the hyper-osmolality group (570 mOsm), the rate of GAG accumula‐ tion/tissue volume wasn't decreased after 2 days of culture when compared with the 370 mOsm group. Values are mean ± standard error. (Scheffe, \*: P<0.05). (B) GAG produced per million cells was significantly increased at 370 and 470 mOsm with time in culture (\*,#: P<0.05, Scheffe between between 2 and 6 days)] This was the highest in the 370 mOsm group and the lowest in the hypo-osmolality group (270 mOsm) decreased after 6 days of culture. In the hyperosmolality group (470 and 570 mOsm), the rate of total GAG produced per million cells wasn't decreased after 2 days of culture when compared with the 370 mOsm group. (Reproduced with permission from Negoro K, Kobayashi S, et al. Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three

In this study, the chondrocytes produced the highest amount of GAG in the osmolality condition of 370 mOsm after 2 and 6 days of culture. The amount of GAG production was obviously lower in the low osmolality cultures than in the culture at the optimal osmolarity close to that in the normal cartilage. On electron microscopy of chondrocytes cultured under varying levels of osmolality, cells under 370 mOsm generally showed normal nuclei and cytoplasm, while cells under hypo-osmolality presented oncotic changes, with cellular swelling and destructed organelles of the cytoplasm. On the other hand, cells cultured under hyper-osmolality were reduced in size and some cells underwent apoptosis. Manjo & Joris (1995) reported oncosis is a form of cell death accompanied by cellular swelling, organelle swelling, blebbing, and increase membrane permeability. They also showed that necrosis can occur after both forms (oncosis and apoptosis) of cell death. Therefore, its mechanism is based on failure of the ionic pumps of the plasma membrane induced by the changes of extracellular osmotic environment. Thus, our physiological and morphological study showed the articular chondrocytes is unable to adjust for such non-physiological conditions lasting for a long time and this phenomenon plays a critical role in the development of cartilage degeneration and

dimensional culture system. Clin Exp Rheumatol 26: 527-533,2008).

552 Regenerative Medicine and Tissue Engineering

resultant OA.

The osmotic pressure in the cartilage tissues is obviously higher than the plasma osmolarity (about 280 mOsm), and chondrocytes exist in the extracellular environment different from that of other tissues. This study indicated that adjustment of osmolality is very important for the culture of chondrocytes. At cell densities found *in vivo* (standard conditions) in the cartilage tissue viz, 4 million cells/ml and GAG concentration in beads cultures was 0.298 mg/ml at 370 mOsm in 6 days. Assuming that the initial production rate is maintained and that there is no loss of GAG, it is calculated that > 1000 days of culture is necessary to produce a GAG concentration equal to the in vivo GAG concentration of 7% per wet weight (viz. 70 mg/ml). That is, it is suggested that chondrocytes need to be cultivated at the cell density of 4 x 106 cells/ml for more than 1 year in order to construct cartilage tissue in the GAG concentration of about 70 - 100 mgs/ml, which is equal to the GAG concentration in the normal cartilage tissues, using cell culture technology.
