**Abstract**

This research study examines the effects of process parameters on fabricated triangular micro holes using micro-EDM (Micro Electrical Discharge Machining). The micro-EDM process utilized a 400 μm tungsten carbide electrode shaped as an equilateral triangle. The machining parameter assessment involved measuring errors in the corner radius and the included angle of the microcavity. The corner radius of the triangular microcavity ranged from 5 to 15 μm. Simultaneously, the included angle varied between 61 and 64°. These variations were attributed to adjustments in capacitance, voltage, and feed settings, which spanned from 1 nF, 90 V, and 5 μm to 100 nF, 130 V, and 15 μm, respectively. Statistical analysis using ANOVA revealed that voltage and capacitance significantly impacted the alterations in corner radii and included the angle of the manufactured triangular microcavities. FESEM is used to analyze the recast layer formation on the edges of the microcavity. It will help to decrease the error in fabricating triangular cavities using micro EDM.

**Keywords:** advanced manufacturing process, micromachining, micro EDM, triangular microcavity, ANOVA


**2. Changes of the extracellular environment in the articular cartilage with age**

Unlike other connective tissues, articular cartilage represents avascular tissue. The chondro‐ cytes may receive nutrients via the vascular system under subchondral bone as well as via the synovial fluid and are supplied by diffusion, helped by the pumping action generated by compression of the articular cartilage or flexion of the elastic cartilage (Brower, et al. 1962, Mankin 1963, Maroudas, et al. 1968, Hodge & McKibbin 1969, McKibbin & Holdsworth 1966, O'Hara et al. 1990). Nutritional supply to the cartilaginous tissue is affected by the architecture of the vascular system and the porosity in the subchondral bone (Fig.1A). Nutrients move from the vascular systems under subchondral bone that supply the cartilaginous tissue, through the subchondral bone and the dense matrix of the cartilaginous tissue, to the chondrocytes. Its limits transport of large molecules into and out of the cartilaginous tissue. For small solutes such as glucose, lactate acid, and oxygen, both experimental and modeling studies have shown that solute transport is accomplished mainly by diffusion (Mauck et al. 2003a), hence, the movement of fluid in and out of the cartilaginous tissue as a result of the diurnal loading pattern has little direct influence on transport. Gradients in the concentration arise depending on the balance between the rate of supply of glucose or oxygen from the blood supply to the cells and the rate of cellular consumption (Stockwell, 1991, Hall, et al. 1993, Lee & Urban, 1997, 2002).

The cartilaginous tissue is avascular, and the metabolic activity of its cells is regulated by various factors in the extracellular matrix, such as oxygen concentration (Lane et al, 1977, Lee & Urban, 1997, 2002), extracellular osmolality (Urban & Bayliss, 1989, Urban et al., 1993, Bush & Hall, 2001, Palmer et al., 2001, Erickson et al., 2001, Bush et al., 2005, Negoro, et al., 2008), pH (Wilkins & Hall, 1995, Gray et al.,1988), mechanical stress (Gray et al. 1989, Urban 1994, 2000), and various growth factors (Morales & Roberts, 1988, Luyten et al, 1988, Morales, 1994, Van Osch, et al. 1998, Huck, 2001). The cell density of the normal human cartilaginous tissue is 2– 4 x 106 cells/mL, and the extracellular environment differs markedly from that of other tissues, with an oxygen saturation of 1–6%, pH of 6.8–7.1, and extracellular osmolality of 350–450mOsm. So, the cartilaginous extracellular environment is relatively hypoxic, unlike the case of other tissues. And also, the articular chondrocytes are exposed to a unique osmotic environment, which varies throughout the depth of cartilage, and in response to mechanical loading or pathological conditions. It is said that such a harsh environment suppresses chondrocyte differentiation and maintains the nature characteristic to chondrocytes. Thus, compared to other connective tissues, cartilage grows and repairs more slowly.

strength of cartilage tissues strongly depends on the density of aggrecan (Kempson et al., 1970, Maroudas, 1979, Maroudas & Bannon 1981). Therefore, in order to produce cartilage tissues that can tolerate mechanical force of about 10-20 Mpa using the tissue engineering technology, it is necessary to generate sufficient PG (Hodge et al., 1986). PG/GAG generation depends on the amount of GAG production, the capacity of GAG retention in the tissues, and the concentration of cells (Kobayashi et al., 2008). There is now an increasing interest in developing biological methods of cartilage repair for these disorders with attainment of the correct biomechanical properties critical for success (Brittberg et al. 1994, Minas, 2001, Risbud & Sittinger 2002, Schaefer et al. 2002 Ochi et al. 2002, Robert et al. 2003). The stiffness of cartilaginous tissues is thus strongly dependent on aggrecan content. Therefore, one of the targets of successful repair is thus that GAG concentration of the tissue-engineered construct should approach that of the native cartilage. First of all, it is important to establish the optimum culture conditions for the generation of cartilaginous tissues. In this study, we examined how physiological levels of extracellular osmolality and cell density influence PG accumulation in chondrocytes in a three-dimensional culture system. And also, we evaluated the influence of transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2), which are involved on the metabolism of PGs by cartilage cells cultured under low-osmotic conditions.

**2. Changes of the extracellular environment in the articular cartilage with**

Unlike other connective tissues, articular cartilage represents avascular tissue. The chondro‐ cytes may receive nutrients via the vascular system under subchondral bone as well as via the synovial fluid and are supplied by diffusion, helped by the pumping action generated by compression of the articular cartilage or flexion of the elastic cartilage (Brower, et al. 1962, Mankin 1963, Maroudas, et al. 1968, Hodge & McKibbin 1969, McKibbin & Holdsworth 1966, O'Hara et al. 1990). Nutritional supply to the cartilaginous tissue is affected by the architecture of the vascular system and the porosity in the subchondral bone (Fig.1A). Nutrients move from the vascular systems under subchondral bone that supply the cartilaginous tissue, through the subchondral bone and the dense matrix of the cartilaginous tissue, to the chondrocytes. Its limits transport of large molecules into and out of the cartilaginous tissue. For small solutes such as glucose, lactate acid, and oxygen, both experimental and modeling studies have shown that solute transport is accomplished mainly by diffusion (Mauck et al. 2003a), hence, the movement of fluid in and out of the cartilaginous tissue as a result of the diurnal loading pattern has little direct influence on transport. Gradients in the concentration arise depending on the balance between the rate of supply of glucose or oxygen from the blood supply to the cells and the rate of cellular consumption (Stockwell, 1991, Hall, et al. 1993, Lee & Urban,

**age**

544 Regenerative Medicine and Tissue Engineering

1997, 2002).

The articular cartilage is essential for absorbing shock and maintaining normal joint environ‐ ment, and, regardless of the cause, degeneration of articular cartilage can result in irreversible osteoarthritis. Osteoarthritis is commonly referred to as wear-and-tear arthritis. There are other parts of joint anatomy, like subchondral bone, that play a significant role in osteoarthritis. Subchondral bone is the layer of bone just below the cartilage. The subchondral bone does not normally provide a barrier to diffusion. In aging and degeneration, however, the subchondral bone tends to calcify by unknown mechanisms and the apparent permeability of this plate decreases with age, as the subchondral bone becomes more sclerotic (Lane, et al., 1977, Lane, & Villacin, 1980, Botter et al., 2011). The consequent fall in supply of nutrients to the chondro‐ cytes inhibits their ability to synthesize and maintain the matrix and even leads to cell death (Kühn, 2004). In osteoarthritis, subchondral bone becomes thicker than usual (Fig1B). Adult hyaline articular cartilage is progressively mineralized at the junction between cartilage and bone. It is then termed articular calcified cartilage. A mineralization front advances through the base of the hyaline articular cartilage at a rate dependent on cartilage load and shear stress. Intermittent variations in the rate of advance and mineral deposition density of the mineral‐ izing front, lead to multiple "tidemarks" in the articular calcified cartilage. Adult articular calcified cartilage is penetrated by vascular buds, and new bone produced in the vascular space in a process similar to endochondral ossification at the physis. A cement line demarcates articular calcified cartilage from subchondral bone. Vascular channels connect the marrow spaces of trabecular bone with the calcified cartilage layer, thus nourishing the deeper cartilage layers that cannot be nourished by synovial fluid (Duncan, 1987, Milz & Putz, 1994). These vascular channels also nourish osteocytes in the subchondral bone plate, unlike osteocytes in trabecular bone, which receive nourishment from marrow tissue. In aging, however, the subchondral bone tends to calcify by unknown mechanisms (Fig.9B). This tide mark (calcifi‐ cation) acts as a barrier to nutrients transport and is thought to be a major factor in the development of osteoarthritis. Cellular parameters are very important in regulating nutrient 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 appropriate ionic and osmotic environment for chondrocytes.

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.