BACKGROUND
osteoarthritis  is a chronic and multifaceted degenerative joint disease in which the articular cartilage and the surrounding extracellular matrix  are destroyed  <cit> .
it is associated with increasing age because the articular cartilage of the joints may degrade with continual wear.
an imbalance between the repair and degradation of the cartilage may disrupt the collagen matrix, resulting in oa.
non-steroidal anti-inflammatory drugs, calcitonin, and glucosamine have been used to treat oa  <cit> .
however, these agents either have serious side effects or may not be ideal for long-term therapy.
recent studies of oa therapeutics have focused primarily on the development of disease-modifying osteoarthritis drugs  and connective tissue structure-modifying agents   <cit> .
doxycycline  has been used to treat the symptoms of oa  <cit> .
the synthesis of inducible nitric oxide synthase is inhibited by dox, which suppresses the secretion of matrix metalloproteinases  by chondrocytes, thus relieving the degradation of type ii collagen and aggrecan.
in addition, dox significantly suppresses the production of inflammatory cytokines, such as interleukin- <dig>  and interleukin- <dig>  which inhibits inflammation in oa synovial cells and chondrocytes  <cit> .
in an anterior-cruciate-ligament rupture-induced spontaneous oa model, dox significantly improved the structure of the subchondral bone  <cit> .
clinical research has also shown that dox can slow the rate of joint-space narrowing in the knees with established oa  <cit> .
thus, the chondroprotective effects of the suppression of catabolic cytokine cascades by dox treatment may represent the ideal properties of both dmoad- and ctsma-based therapies for oa.
hyaluronic acid  occurs naturally in the ecm and synovial fluid.
imbalances in ha stability can result in the development of oa, and the joints of oa patients have been shown to contain shorter ha fragments than those found in normal joints  <cit> .
the clinical outcomes of intra-articular ha and derived products had been critically reviewed, and proven to be an effective, safe, and tolerable treatment for knee oa  <cit> .
intra-articular injections of ha and other lubricating substances have been shown to relieve oa symptoms by alleviating pain and irritation  <cit> .
inflammation reduces the viscoelasticity of synovial fluid, and intra-articular injections of ha can compensate for the loss of joint lubrication by adhering to the cartilage surfaces and protecting them from damage  <cit> .
it has been reported that nonmodified ha only with a half-life of 10–13 h, while chemically modified ha product, such as hylan g-f  <dig>  was lasting to  <dig>  ±  <dig>  days  <cit> .
the rapid clearance and elimination of ha intra-articular injection may limit its clinical usage; therefore, the improvements of longer residence time may extend the therapeutic benefit and clinical applications.
we hypothesized that the combination of ha and dox in a hydrogel  <cit>  might produce additive effects in oa therapy through the anti-inflammatory and analgesic effects of dox and ha, respectively, and the increased viscoelasticity of the synovial fluid resulting from the high-molecular-weight hydrogel polymer.
in addition, the slow release of dox from the hydrogel polymer may sustain its therapeutic effects.
we produced an injectable ha-dox hydrogel, and evaluated its efficacy as an intra-articular treatment in a rabbit model of oa.
RESULTS
rheological characteristics of the ha-dox hydrogels
the mixture of ha, dox, and zncl in an aqueous solution produced a thermo-reversible, water-soluble hydrogel.
because it had been previously reported that the higher-molecular-weight, more viscoelastic hylan g-f  <dig> has significantly greater pain-relieving properties than does the lower-molecular-weight, less-viscoelastic ha  <cit> , the rheological characteristics  of the ha-dox hydrogel and an ha-dox mixture without zinc cations were assessed over a range of dox concentrations .
note:  <dig>  hz is the approximate frequency corresponding to the movement of the knee joint in walking; g’, dynamic elastic modulus; g”, dynamic viscous modulus.
hydrogel, containing 10 mg/ml ha and indicated concentrations of dox with zn2+; solution, containing 10 mg/ml ha and indicated concentrations of dox without zn2+.
#concentration of ha = 10 mg/ml.
*different concentration of dox  in μg/ml.
<dig>  hz is the approximate frequency corresponding to the movement of the knee joint in running.
in the absence of zinc cations, increasing concentrations of dox caused progressive decreases in both the g’ and the g” as the ha and dox precipitated because of charge interactions.
compared with the ha, both the g’ and g” were higher for the ha-dox hydrogel, with the g’ and the g” increasing with dox concentrations in the presence of zinc cations, indicating that ionic bonding between the ha and the dox molecules had occurred within the high-mw polymer.
moreover, as the oscillating frequency decreased, both the g’ and the g” decreased more slowly as the dox concentration was increased, which further indicated that a high degree of dox cross-linking had occurred.
we concluded that the viscoelasticity of the ha-dox hydrogel was comparable to or greater than that of the ha.
ha-dox hydrogel exhibited low cytotoxicity in vitro
as shown in figure 1a, sw <dig> cell viability was not noticeably influenced by  <dig>  to 25 μg/ml dox, whereas 50 μg/ml dox significantly inhibited the survival of sw <dig> cells.
treatments of sw <dig> cells with  <dig> ,  <dig> ,  <dig> , and  <dig>  mg/ml ha for 24 h showed no cytotoxicity .
treatments with ha-dox hydrogels containing 4 mg/ml ha and  <dig> ,  <dig> ,  <dig> , or  <dig>  μg/ml dox for 24 h did not reduce sw <dig> cell viability or alter cell morphology .
intra-articular ha-dox hydrogel injections reduced pain
following the surgical induction of oa, the percentage weight distribution of the left-hind paw decreased significantly in each group .
the percentage weight distribution values for both the nt and the dox groups decreased over the course of the experiment.
in the ha and ha-dox hydrogel groups, the percentage weight distributions showed gradual and significant increases on  <dig>   <dig>  and 13 d after surgery, compared with the nt and dox groups , indicating that the intra-articular injection of the ha and the ha-dox hydrogel treatments exhibited analgesic effects.
intra-articular ha-dox hydrogel injections reduced oa pathology
the macroscopic examination of the specimens showed that the articular cartilage was rough and dull on both the femoral and tibial surfaces.
the most remarkable damage in each group occurred at the femoral condyle, the femoral trochlea, and the tibial plateau.
the nt group had the highest lesion scores for all parameters among the various treatment groups, and the ha-dox hydrogel group had significantly lower scores for all parameters .
although the differences between the ha and the ha-dox hydrogel groups were not statistically significant for all oa features examined, lower lesion scores for the loss of the superficial layer of the femur, the presence of fibrillation, and the presence of osteophytes of the femur and the tibia were recorded for the ha-dox hydrogel group.
overall, the intra-articular injection of the ha-dox hydrogel significantly inhibited the progression of oa in the rabbit model.
the microscopic histological examination showed that the intra-articular injection of the ha-dox hydrogel reduced the loss of chondrocytes at the femoral condyles  and at the tibial plateau .
treatment with either the ha-dox hydrogel or the ha attenuated lesion formation.
the nt group had many chondrocytes, whereas the dox group had few.
the clone parameter  revealed a typical feedback situation.
the ha-dox hydrogel group had the fewest chondrocyte clones, whereas the dox, ha, and nt groups showed significant cloning of chondrocytes.
the nt group showed the lowest loss of proteoglycan.
both the ha and the ha-dox hydrogel groups showed reduced losses of the superficial layer, compared with the dox and nt groups .
similar results were observed for ulceration, fibrillation, osteophytes, fissures, and disorganization of chondrocytes, with the ha and the ha-dox hydrogel groups displaying significantly lower scores for most of the parameters.
overall, the ha-dox hydrogel treatment resulted in the greatest reductions in oa pathology.
discussion
in our study, the ha-dox hydrogel was formed by polymerizing electrostatic interactions between ha and dox molecules, followed by cross-linking through the phenolic moieties of the immobilized dox by zinc-mediated chelation.
increasing dox concentrations in the hydrogel exhibited increased g’ and g” values for the polymer, with the higher dox concentrations resulting in a more highly cross-linked structure that displayed greater viscosity and viscoelasticity.
our results show that the enhanced rheological properties of the ha-dox hydrogel were beneficial for intra-articular applications in oa therapy.
when applied intra-articularly, ha covers the surface of the cartilage, acting as a cushion that absorbs pressure and vibration and prevents further erosion.
the highly viscous ha has been used in intra-articular injections to reverse the loss of viscosity and viscoelasticity of the synovial fluid resulting from inflammation and physical wear  <cit> .
our data show that the ha-dox hydrogel possesses higher viscosity and viscoelasticity than those of ha alone, suggesting that it may provide better chondroprotective effects .
the cytotoxicity evaluation showed that treatment with high concentrations of dox alone  was toxic to the sw <dig> chondrosarcoma cells.
however, the ha-dox hydrogel displayed significantly lower cytotoxicity, compared with similar concentrations of dox .
these results suggest that the ha-dox hydrogel may maintain protective effects for longer periods than those of either ha or dox used alone.
intra-articular application of ha promotes the nutrient transport and waste excretion functions of synovial fluid, and covers the articular surfaces to protect the cartilage.
the adherence of ha to articular surfaces has been reported to protect nerve endings that may be exposed by thinner, degraded cartilage  <cit> .
the pain-relieving property of ha is an important disease-modifying quality for clinical purposes.
thus, the increase in viscosity and viscoelasticity of the ha-dox hydrogel indicates that it should produce similar effects.
in a prospective cohort study to evaluate pain and functional outcomes from oa patients with intra-articular ha injection over 6 months, ha was associated with lower functional pain severity, which represented an increase in the quality of the movement and functional activity  <cit> .
another study had also reported the mid-term efficacy  of intra-articular ha injection improve the joint structure and relieve pain in patients with knee oa  <cit> .
in our in vivo experiments, the percentage weight distributions of the injured hind paw were used as an indicator of the analgesic effects of the treatments .
the results showed a significant analgesic effect following intra-articular injection of the ha and the ha-dox hydrogel, compared with the dox and nt treatments, on days  <dig>   <dig>  and  <dig> following the induction of oa and the initiation of treatment.
this indicates the ha-dox hydrogel can still retain the pain-relieving property of ha; however, its long-term effect of pain-reliving need be further studied.
the macroscopic examination revealed that both the ha and the ha-dox hydrogel treatments diminished oa features, compared with the nt and dox treatments.
moreover, the ha-dox hydrogel group exhibited greater therapeutic effects than those observed in the ha group.
however, intra-articular injection of dox alone did not abrogate the oa pathology, compared with the results observed in the nt group .
histopathological findings  mirrored the results of the macroscopic evaluation and pain assessment.
both the ha and the ha-dox hydrogel treatments reduced cartilage degradation at the femoral condyles and the tibial plateau, and effectively reduced the loss of the superficial layer, ulceration, the production of osteophytes, the creation of fissures, and the disorganization of cartilage, compared with the nt and dox treatments .
as the results of macroscopic examination, the chondroprotective effects of ha-dox hydrogel treatment were superior to those of the ha treatment.
the therapeutic treatments had started immediately following surgery in this study that may limit the therapeutic outcomes interpreting.
intra-articular hylan g-f  <dig> injection in 4 weeks post-operatively to the same model had also been reported the protective effects in maintains cartilage integrity and decreases osteophyte formation  <cit> .
this may imply the potential of the ha-dox hydrogel in application to oa models with the onset of symptoms.
future studies with different therapeutic regimens of ha-dox hydrogel could provide additional support to our current findings.
the loss of chondrocytes and of proteoglycan scores for the nt group was lower than that of the ha group and that of the ha and ha-dox hydrogel groups, respectively.
this may result in a feedback phenomenon, in which a greater amount of chondrocytes produced comparatively greater amounts of proteoglycan.
however, since the nt group presented similar proteoglycan level to normal groups, it still cannot rule out the possibility of the adversary effects of proteoglycan depletion following the treatments.
as erosion and loss of cartilage occurred, the remaining chondrocytes secreted more glycoprotein for bone regeneration, resulting in the production of osteophytes.
the nt group had the highest score of osteophytes; this may further support the involvement of such feedback.
the clone parameter assessed the aggregation of chondrocytes, which may also result from the feedback phenomenon.
aggregation results from abnormal cell proliferation following cartilage destruction or chondrocyte perturbations.
thus, clones can serve as signs of altered cartilage.
however, the reliability of this inference has not been thoroughly established.
in addition to ha-dox hydrogel, several different ha-derived combination compounds were recently reported the therapeutic effects by intra-articular injection in rabbit oa model.
celecoxib-loaded liposomes embedded in ha gel combination was more effective than a single drug in pain control and cartilage protection  <cit> .
intra-articular injection of collagen tripeptide, and collagen tripeptide and ha mixtures seemed to be effective for the initiation period of cartilage degeneration partly by promotion of type ii collagen synthesis and prevention of proteoglycan loss  <cit> .
these findings suggest the developing potential of ha-derived combination compounds in the oa therapy.
it is unlikely that the beneficial effects of the ha-dox hydrogel treatment can be attributed to a temporary enhancement of the viscoelasticity and viscosity of the synovial fluid.
based on the demonstrated anti-inflammatory effects of dox in clinical applications, the ha-dox hydrogel therapy possibly produced disease-modifying biological actions that influenced the progression of oa in the rabbit model.
thus, the ha-dox hydrogel represents an ideal dmoad and ctsma.
the long-term therapeutic effects of intra-articular ha-dox hydrogel treatment in chronic oa warrants further investigation.
CONCLUSIONS
the ha-dox hydrogel, composed of a polymer of ha and dox with zinc cations, possessed higher viscosity and viscoelasticity, and provided better chondroprotective effects than those of an ha alone.
therefore, the injectable ha-dox hydrogel may represent a desirable dmoad for oa therapy.

 osteoarthritis is a chronic and multifaceted degenerative joint disease in which the articular cartilage and the surrounding extracellular matrix are destroyed. it is associated with increasing age because the articular cartilage of the joints may degrade with continual wear. an imbalance between the repair and degradation of the cartilage may disrupt the collagen matrix, resulting in oa. non-steroidal anti-inflammatory drugs, calcitonin, and glucosamine have been used to treat oa. however, these agents either have serious side effects or may not be ideal for long-term therapy. recent studies of oa therapeutics have focused primarily on the development of disease-modifying osteoarthritis drugs and connective tissue structure-modifying agents. doxycycline has been used to treat the symptoms of oa. the synthesis of inducible nitric oxide synthase is inhibited by dox, which suppresses the secretion of matrix metalloproteinases by chondrocytes, thus relieving the degradation of type ii collagen and aggrecan. in addition, dox significantly suppresses the production of inflammatory cytokines, such as interleukin- and interleukin- which inhibits inflammation in oa synovial cells and chondrocytes. in an anterior-cruciate-ligament rupture-induced spontaneous oa model, dox significantly improved the structure of the subchondral bone. clinical research has also shown that dox can slow the rate of joint-space narrowing in the knees with established oa. thus, the chondroprotective effects of the suppression of catabolic cytokine cascades by dox treatment may represent the ideal properties of both dmoad- and ctsma-based therapies for oa. hyaluronic acid occurs naturally in the ecm and synovial fluid. imbalances in ha stability can result in the development of oa, and the joints of oa patients have been shown to contain shorter ha fragments than those found in normal joints. the clinical outcomes of intra-articular ha and derived products had been critically reviewed, and proven to be an effective, safe, and tolerable treatment for knee oa. intra-articular injections of ha and other lubricating substances have been shown to relieve oa symptoms by alleviating pain and irritation. inflammation reduces the viscoelasticity of synovial fluid, and intra-articular injections of ha can compensate for the loss of joint lubrication by adhering to the cartilage surfaces and protecting them from damage. it has been reported that nonmodified ha only with a half-life of 10–13 h, while chemically modified ha product, such as hylan g-f was lasting to  ±   days. the rapid clearance and elimination of ha intra-articular injection may limit its clinical usage; therefore, the improvements of longer residence time may extend the therapeutic benefit and clinical applications. we hypothesized that the combination of ha and dox in a hydrogel might produce additive effects in oa therapy through the anti-inflammatory and analgesic effects of dox and ha, respectively, and the increased viscoelasticity of the synovial fluid resulting from the high-molecular-weight hydrogel polymer. in addition, the slow release of dox from the hydrogel polymer may sustain its therapeutic effects. we produced an injectable ha-dox hydrogel, and evaluated its efficacy as an intra-articular treatment in a rabbit model of oa.  rheological characteristics of the ha-dox hydrogels the mixture of ha, dox, and zncl in an aqueous solution produced a thermo-reversible, water-soluble hydrogel. because it had been previously reported that the higher-molecular-weight, more viscoelastic hylan g-f has significantly greater pain-relieving properties than does the lower-molecular-weight, less-viscoelastic ha, the rheological characteristics of the ha-dox hydrogel and an ha-dox mixture without zinc cations were assessed over a range of dox concentrations. note:  hz is the approximate frequency corresponding to the movement of the knee joint in walking; g’, dynamic elastic modulus; g”, dynamic viscous modulus. hydrogel, containing 10 mg/ml ha and indicated concentrations of dox with zn2+; solution, containing 10 mg/ml ha and indicated concentrations of dox without zn2+. #concentration of ha = 10 mg/ml. *different concentration of dox in μg/ml.  hz is the approximate frequency corresponding to the movement of the knee joint in running. in the absence of zinc cations, increasing concentrations of dox caused progressive decreases in both the g’ and the g” as the ha and dox precipitated because of charge interactions. compared with the ha, both the g’ and g” were higher for the ha-dox hydrogel, with the g’ and the g” increasing with dox concentrations in the presence of zinc cations, indicating that ionic bonding between the ha and the dox molecules had occurred within the high-mw polymer. moreover, as the oscillating frequency decreased, both the g’ and the g” decreased more slowly as the dox concentration was increased, which further indicated that a high degree of dox cross-linking had occurred. we concluded that the viscoelasticity of the ha-dox hydrogel was comparable to or greater than that of the ha. ha-dox hydrogel exhibited low cytotoxicity in vitro as shown in figure 1a, sw cell viability was not noticeably influenced by to 25 μg/ml dox, whereas 50 μg/ml dox significantly inhibited the survival of sw cells. treatments of sw cells with,,, and  mg/ml ha for 24 h showed no cytotoxicity. treatments with ha-dox hydrogels containing 4 mg/ml ha and,,, or  μg/ml dox for 24 h did not reduce sw cell viability or alter cell morphology. intra-articular ha-dox hydrogel injections reduced pain following the surgical induction of oa, the percentage weight distribution of the left-hind paw decreased significantly in each group. the percentage weight distribution values for both the nt and the dox groups decreased over the course of the experiment. in the ha and ha-dox hydrogel groups, the percentage weight distributions showed gradual and significant increases on and 13 d after surgery, compared with the nt and dox groups, indicating that the intra-articular injection of the ha and the ha-dox hydrogel treatments exhibited analgesic effects. intra-articular ha-dox hydrogel injections reduced oa pathology the macroscopic examination of the specimens showed that the articular cartilage was rough and dull on both the femoral and tibial surfaces. the most remarkable damage in each group occurred at the femoral condyle, the femoral trochlea, and the tibial plateau. the nt group had the highest lesion scores for all parameters among the various treatment groups, and the ha-dox hydrogel group had significantly lower scores for all parameters. although the differences between the ha and the ha-dox hydrogel groups were not statistically significant for all oa features examined, lower lesion scores for the loss of the superficial layer of the femur, the presence of fibrillation, and the presence of osteophytes of the femur and the tibia were recorded for the ha-dox hydrogel group. overall, the intra-articular injection of the ha-dox hydrogel significantly inhibited the progression of oa in the rabbit model. the microscopic histological examination showed that the intra-articular injection of the ha-dox hydrogel reduced the loss of chondrocytes at the femoral condyles and at the tibial plateau. treatment with either the ha-dox hydrogel or the ha attenuated lesion formation. the nt group had many chondrocytes, whereas the dox group had few. the clone parameter revealed a typical feedback situation. the ha-dox hydrogel group had the fewest chondrocyte clones, whereas the dox, ha, and nt groups showed significant cloning of chondrocytes. the nt group showed the lowest loss of proteoglycan. both the ha and the ha-dox hydrogel groups showed reduced losses of the superficial layer, compared with the dox and nt groups. similar results were observed for ulceration, fibrillation, osteophytes, fissures, and disorganization of chondrocytes, with the ha and the ha-dox hydrogel groups displaying significantly lower scores for most of the parameters. overall, the ha-dox hydrogel treatment resulted in the greatest reductions in oa pathology. discussion in our study, the ha-dox hydrogel was formed by polymerizing electrostatic interactions between ha and dox molecules, followed by cross-linking through the phenolic moieties of the immobilized dox by zinc-mediated chelation. increasing dox concentrations in the hydrogel exhibited increased g’ and g” values for the polymer, with the higher dox concentrations resulting in a more highly cross-linked structure that displayed greater viscosity and viscoelasticity. our results show that the enhanced rheological properties of the ha-dox hydrogel were beneficial for intra-articular applications in oa therapy. when applied intra-articularly, ha covers the surface of the cartilage, acting as a cushion that absorbs pressure and vibration and prevents further erosion. the highly viscous ha has been used in intra-articular injections to reverse the loss of viscosity and viscoelasticity of the synovial fluid resulting from inflammation and physical wear. our data show that the ha-dox hydrogel possesses higher viscosity and viscoelasticity than those of ha alone, suggesting that it may provide better chondroprotective effects. the cytotoxicity evaluation showed that treatment with high concentrations of dox alone was toxic to the sw chondrosarcoma cells. however, the ha-dox hydrogel displayed significantly lower cytotoxicity, compared with similar concentrations of dox. these results suggest that the ha-dox hydrogel may maintain protective effects for longer periods than those of either ha or dox used alone. intra-articular application of ha promotes the nutrient transport and waste excretion functions of synovial fluid, and covers the articular surfaces to protect the cartilage. the adherence of ha to articular surfaces has been reported to protect nerve endings that may be exposed by thinner, degraded cartilage. the pain-relieving property of ha is an important disease-modifying quality for clinical purposes. thus, the increase in viscosity and viscoelasticity of the ha-dox hydrogel indicates that it should produce similar effects. in a prospective cohort study to evaluate pain and functional outcomes from oa patients with intra-articular ha injection over 6 months, ha was associated with lower functional pain severity, which represented an increase in the quality of the movement and functional activity. another study had also reported the mid-term efficacy of intra-articular ha injection improve the joint structure and relieve pain in patients with knee oa. in our in vivo experiments, the percentage weight distributions of the injured hind paw were used as an indicator of the analgesic effects of the treatments. the results showed a significant analgesic effect following intra-articular injection of the ha and the ha-dox hydrogel, compared with the dox and nt treatments, on days and following the induction of oa and the initiation of treatment. this indicates the ha-dox hydrogel can still retain the pain-relieving property of ha; however, its long-term effect of pain-reliving need be further studied. the macroscopic examination revealed that both the ha and the ha-dox hydrogel treatments diminished oa features, compared with the nt and dox treatments. moreover, the ha-dox hydrogel group exhibited greater therapeutic effects than those observed in the ha group. however, intra-articular injection of dox alone did not abrogate the oa pathology, compared with the results observed in the nt group. histopathological findings mirrored the results of the macroscopic evaluation and pain assessment. both the ha and the ha-dox hydrogel treatments reduced cartilage degradation at the femoral condyles and the tibial plateau, and effectively reduced the loss of the superficial layer, ulceration, the production of osteophytes, the creation of fissures, and the disorganization of cartilage, compared with the nt and dox treatments. as the results of macroscopic examination, the chondroprotective effects of ha-dox hydrogel treatment were superior to those of the ha treatment. the therapeutic treatments had started immediately following surgery in this study that may limit the therapeutic outcomes interpreting. intra-articular hylan g-f injection in 4 weeks post-operatively to the same model had also been reported the protective effects in maintains cartilage integrity and decreases osteophyte formation. this may imply the potential of the ha-dox hydrogel in application to oa models with the onset of symptoms. future studies with different therapeutic regimens of ha-dox hydrogel could provide additional support to our current findings. the loss of chondrocytes and of proteoglycan scores for the nt group was lower than that of the ha group and that of the ha and ha-dox hydrogel groups, respectively. this may result in a feedback phenomenon, in which a greater amount of chondrocytes produced comparatively greater amounts of proteoglycan. however, since the nt group presented similar proteoglycan level to normal groups, it still cannot rule out the possibility of the adversary effects of proteoglycan depletion following the treatments. as erosion and loss of cartilage occurred, the remaining chondrocytes secreted more glycoprotein for bone regeneration, resulting in the production of osteophytes. the nt group had the highest score of osteophytes; this may further support the involvement of such feedback. the clone parameter assessed the aggregation of chondrocytes, which may also result from the feedback phenomenon. aggregation results from abnormal cell proliferation following cartilage destruction or chondrocyte perturbations. thus, clones can serve as signs of altered cartilage. however, the reliability of this inference has not been thoroughly established. in addition to ha-dox hydrogel, several different ha-derived combination compounds were recently reported the therapeutic effects by intra-articular injection in rabbit oa model. celecoxib-loaded liposomes embedded in ha gel combination was more effective than a single drug in pain control and cartilage protection. intra-articular injection of collagen tripeptide, and collagen tripeptide and ha mixtures seemed to be effective for the initiation period of cartilage degeneration partly by promotion of type ii collagen synthesis and prevention of proteoglycan loss. these findings suggest the developing potential of ha-derived combination compounds in the oa therapy. it is unlikely that the beneficial effects of the ha-dox hydrogel treatment can be attributed to a temporary enhancement of the viscoelasticity and viscosity of the synovial fluid. based on the demonstrated anti-inflammatory effects of dox in clinical applications, the ha-dox hydrogel therapy possibly produced disease-modifying biological actions that influenced the progression of oa in the rabbit model. thus, the ha-dox hydrogel represents an ideal dmoad and ctsma. the long-term therapeutic effects of intra-articular ha-dox hydrogel treatment in chronic oa warrants further investigation.  the ha-dox hydrogel, composed of a polymer of ha and dox with zinc cations, possessed higher viscosity and viscoelasticity, and provided better chondroprotective effects than those of an ha alone. therefore, the injectable ha-dox hydrogel may represent a desirable dmoad for oa therapy. 
overall, from both macroscopic and microscopic data of this study indicate the injectable ha-dox hydrogels presented as a long-lasting pharmacotherapeutic agent to apply for oa therapy. we evaluated the therapeutic effects of intra-articular injections of hydrogels containing hyaluronic acid and doxycycline in a rabbit oa model. thirteen week old new zealand white rabbits undergone a partial meniscectomy and unilateral fibular ligament transection were administered with either normal saline, ha, dox or ha-dox hydrogels on day and 12; animals were also examined the pain assessment in every three days. the degree of pain was significantly attenuated after day post-treatment with both ha and ha-dox hydrogels. in addition, ha-dox hydrogels also ameliorated the progression of oa by protecting the injury of articular cartilage layer and restoring the elastoviscosity. in macroscopic appearance, ha-dox hydrogel group showed a smoother cartilage surface, no or minimal signs of ulceration, smaller osteophytes, and less fissure formation in compare to ha or dox treatment alone. in the areas with slight oa changes, ha-dox hydrogel group exhibited normal distribution of chondrocytes, indicating the existence of cartilage regeneration.
 osteoarthritis is a common joint disease that causes disabilities in elderly adults. however, few long-lasting pharmacotherapeutic agents with low side effects have been developed to treat oa. we evaluated the therapeutic effects of intra-articular injections of hydrogels containing hyaluronic acid and doxycycline in a rabbit oa model.  thirteen week old new zealand white rabbits undergone a partial meniscectomy and unilateral fibular ligament transection were administered with either normal saline, ha, dox or ha-dox hydrogels on day and 12; animals were also examined the pain assessment in every three days. the joint samples were taken at day post-surgery for further histopathological evaluation. the degree of pain was significantly attenuated after day post-treatment with both ha and ha-dox hydrogels. in macroscopic appearance, ha-dox hydrogel group showed a smoother cartilage surface, no or minimal signs of ulceration, smaller osteophytes, and less fissure formation in compare to ha or dox treatment alone. in the areas with slight oa changes, ha-dox hydrogel group exhibited normal distribution of chondrocytes, indicating the existence of cartilage regeneration. in addition, ha-dox hydrogels also ameliorated the progression of oa by protecting the injury of articular cartilage layer and restoring the elastoviscosity.  overall, from both macroscopic and microscopic data of this study indicate the injectable ha-dox hydrogels presented as a long-lasting pharmacotherapeutic agent to apply for oa therapy. osteoarthritisdisease-modifying osteoarthritis drugsconnective tissue structure-modifying agentshyaluronic aciddoxycycline 