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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: J Orthop Res. 2019 Sep 13;38(1):59–69. doi: 10.1002/jor.24459

In-Vivo Efficacy of Recombinant Human Hyaluronidase (rHuPH20) Injection for Accelerated Healing of Murine Retrocalcaneal Bursitis and Tendinopathy

Sabah N Rezvani 1, Jinnan Chen 2, Jun Li 2, Ron Midura 3, Valbona Cali 3, John D Sandy 4, Anna Plaas 2, Vincent M Wang 1
PMCID: PMC6917826  NIHMSID: NIHMS1048838  PMID: 31478241

Abstract

The deposition of aggrecan/hyaluronan (HA)-rich matrix within the tendon body and surrounding peritenon impede tendon healing and result in compromised biomechanical properties. Hence, the development of novel strategies to achieve targeted removal of the aggrecan-hyaluronan pericellular matrix may be effective in treating tendinopathy. The current study examined the therapeutic potential of a recombinant human hyaluronidase, rHuPH20 for treating murine Achilles tendinopathy. 12-week old C57Bl/6 male mice were injected with two doses of rHuTGF-β1 into the retrocalcaneal bursa (RCB) to induce a combined bursitis and tendinopathy. 24 hours following induction of injury, treatment groups were administered rHu-PH20 Hyaluronidase (rHuPH20, Halozyme Therapeutics) into the RCB. At either 6 hours (acute), 9 days, or 25 days following hyaluronidase treatment, Achilles tendons were analyzed for gene expression, histology and immunohistochemistry, fluorophore assisted carbohydrate electrophoresis (FACE), and biomechanical properties. rHuPH20 treatment was effective, particularly at the acute and 9-day time points, in (a) removing HA deposits from the Achilles tendon and surrounding tissues, (b) improving biomechanical properties of the healing tendon, and (c) eliciting targeted increases in expression of specific cell fate, ECM metabolism, and inflammatory genes. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the RCB and tendon strongly supports the future refinement of injectable glycosidase preparations as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse.

INTRODUCTION

The retrocalcaneal bursa (RCB) is situated anterior to the Achilles tendon and its lateral expansions 1 near the tendon’s calcaneal insertion. The tissues lining the bursal cavity consists of tendinous (Achilles peritenon) posterior wall, fibrocartilaginous anterior wall, and its superior margin is Kager’s fat pad 2, 3 with a synovial type lining. Inflammation of the bursa (retrocalcaneal bursitis) can present with pain and limited function, and can extend to affect both the bone insertion of the Achilles tendon as well as the tendon proper, due to the very close proximity of these structures 4. Active resisted plantar flexion and dorsiflexion are common motions which elicit pain, which may be exacerbated by overuse 5. In the clinical setting, retrocalcaneal bursitis is routinely treated with either corticosteroid injection, non-steroidal anti-inflammatory drug (NSAIDs) administration or activity modification, all directed to managing tendinopathies to protect the tendon from long-term irreversible damage 4. However, anti-inflammatory drugs for tendinopathy show poor-long term outcomes 6, and the limited surgical options involve significant compliance during recovery, a requirement that is often difficult for patients to adhere to, and inadequate healing or wound complications can lead to morbidity of the tissue 7, 8.

A common histopathologic finding in tendinopathies is the deposition of aggrecan/hyaluronan-rich complexes 9, 10 within the tendon body and the surrounding peritenon 11, and these matrix alterations impede fibrogenic healing and result in compromised biomechanical properties 12. Therefore, the development of novel strategies to achieve targeted removal of the aggrecan-hyaluronan pericellular matrix may be effective in treating tendinopathy.

HA accumulation in the tumor microenvironment has been associated with more aggressive malignancy of cancerous tumors and has motivated the development of therapeutics to suppress HA accumulation and/or degrade HA in the tumor stroma 13. While the use of human recombinant hyaluronidases to temporarily remove HA-rich extracellular matrix (ECM) has emerged as a promising therapeutic strategy for reducing HA accumulation in tumors 14–17, it has received very little attention in musculoskeletal applications. Hence, the objective of the current study was to assess the potential of clinical-grade human recombinant hyaluronidase to promote healing of retrocalcaneal bursitis and its associated tendinopathy using a novel murine model.

MATERIALS AND METHODS

Murine Retrocalcaneal Bursitis Model and rHu-PH20 administration

Under IACUC approval, 12-week old C57Bl/6 male mice were injected unilaterally with two doses of 100ng of rHuTGF-β1 (Peptrotech, Inc, Rocky Hill, NJ) into the fat pad and bursa adjacent to the Achilles tendon, at day 0 and day 2. The needle was injected into the Kager’s fat pad on the lateral side of the hindlimb, parallel to the Achilles tendon. The needle (28G) was inserted distally until reaching the posterior aspect of the calcaneus, withdrawing the needle and ensuring the retrocalcaneal bursa was reached (Supplemental Figure 5). Injections were given between 1:00 and 4:00PM and all euthanasia and tissue collections were likewise completed between 1:00 and 4:00PM. No significant changes in body weight or behavior between naïve and treatment groups were observed.

At 24 hours post TGFβ1-injury, 10 μL of 10mg/mL rHu-PH20 Hyaluronidase (rHuPH20, Halozyme Therapeutics, San Diego, CA) in sterile PBS 14, 15 was injected into the retrocalcaneal bursa. The rHu-PH20 formulation used was previously shown not to induce any detectable immune responses when administered in mice 18. Mice euthanized at either 6 hours (acute), 9 days (9d), or 25days (25d) after the second rHuPH20 administration (Supplemental Figure 1). Age-matched un-injured naïve, as well as injured (i.e., TGF-β1 injected) non-treated, groups were also included in the study design.

An additional group of mice was also used to confirm rHuPH20 activity and specificity by substitution of Streptomyces hyaluronidase as injectate. While rHuPH20 efficacy was very similar to that observed for Streptomyces Hyaluronidase (as assessed by IHC and FACE analyses, unpublished data), we did not include this bacterial enzyme in additional studies, since it has limited therapeutic potential, due to potential immune responses in a mammalian host. Details of injection and euthanasia schedules are given in Supplemental Figure 1. Experimental groups, mouse numbers (total for all groups: 493) and outcomes measures are provided in Supplemental Table 1. Briefly, acute, 9d, and 25d post-injury time points were examined, while the experimental groups included “uninjured,” “+TGFβ,” and “+rHuPH20.”

Histology and Immunohistochemistry

Ankle joints (n = 4 per experimental group) were dissected immediately after euthanasia and prepared for histological evaluation as previously described 6. Briefly, tissues were fixed in 10 % formaldehyde for one week, decalcified in EDTA (5% w/v in H2O) for 4 weeks, processed, embedded in paraffin, and cut into sagittal sections of 5 µm thickness through the entire joint. Sections were stained with Safranin-O (Saf-O) for evaluation of tissue responses to injury and rHuPH20 treatment 9. Remaining sections were used for histochemical staining with the following probes: anti-DLS (1 μg/mL 9), followed by biotinylated anti-rabbit IgG as secondary antibody or biotinylated TSG6 (HTI 601, obtained from Halozyme; 0.25 μg/mL). All sections were counterstained with methyl green and assessed by microscopic evaluation. Images from non-immune and Streptomyces Hyaluronidase pre-treatment controls have previously been published as Supplemental Figure 1 of Bitterman et al. 6.

QPCR Gene Expression in Isolated Tendons

Achilles tendons (including peritenon) were isolated immediately following euthanasia, placed in RNA-Later and stored at −20°C until further analyses. RNA was isolated as described previously 10 from each pool of Achilles tendons in each experimental group (Supplemental Table 1). Briefly, tissues were snap-frozen in liquid nitrogen in a Bessman Tissue Pulverizer and extracted in 1mL of Trizol by high-speed vortexing 3x for 60 seconds. RNA was purified using the RNeasy MiniKit (Qiagen, Cat #: 74104, Valencia, CA) with yields of ~350ng (and up to 1697 for acute +rHuPH20) RNA per tendon for all groups. RNA quality (A260:280) was at 1.90–2.15 for all samples. cDNA synthesis was carried out with 2µg of RNA using the RT2 First Strand Kit (Qiagen). RT-qPCR was performed using customized Taqman Array plates (Life Technologies, Carlsbad CA). Gene listing, primer ID numbers and ΔCt values (vs B2m housekeeping gene) for each gene in uninjured tissues are provided in Supplemental Figures 2 and 3. To compare expression levels across experimental groups, fold change (2^-ΔΔCt) was calculated using changes in transcript abundance (ΔCt = Ct for gene of interest minus Ct for the housekeeping gene B2m). Specifically, +TGFβ results were compared to uninjured samples, while +rHuPH20 results were compared to both +TGFβ and uninjured samples in calculating ΔΔCt. An additional control group of mice received an injection of sterile PBS (post-injury), in place of rHuPH20.

Biomechanical testing

Tensile testing and quantification of mechanical properties were carried out as described by Wang et al. 19. Briefly, the murine Achilles/calcaneus complex was dissected from the surrounding tissues, followed by measurements of tendon width and thickness with the assistance of a laser displacement sensor (Model #LK-G82, Keyence, Itasca, IL), from which cross-sectional area (CSA) was computed. The foot was potted in dental cement and the tendon-bone construct was mounted onto a material testing system (MTS Insight 10, Eden Prairie, MN). Tensile testing (in a saline-filled chamber) consisted of preconditioning, stress relaxation test for 15 minutes at 5% strain, followed by a load to failure test at 0.05 mm/sec.

Fluorophore Assisted Carbohydrate Electrophoresis (FACE)

HA and chondroitin sulfate/dermatan sulfate (CS/DS) contents of the Achilles tendon and surrounding peritenon were quantified as previously described 20. Tissue pools (Supplemental Table 1) were digested with 150µL of Proteinase K (10mg/mL in PBS) at 55°C for 18h prior to isolation of GAGs, chondroitinase ABC digestions, fluorotaging, and electrophoretic separation of ΔdiHA, Δdi0S, Δdi6S, and Δdi4S 21, 22. Total HA and CS/DS content was calculated on a per tendon basis. FACE analyses were initially conducted on n=6 or n=8 tendons per pool, to ascertain that the amounts of HA and CS/DS in the digested tissue pools was within the sensitivity range of the FACE assay20. Additional pools were then reduced to n=4 tendons to minimize the number of mice used.

Statistical Methods

All statistical analyses were completed using JMP Pro 14 (SAS, Cary, NC). For gene expression outcome measures, a 1-way ANOVA with Tukey’s post-hoc tests were conducted on ΔCt values to determine significant (p<0.05) differences. Similarly, biomechanical and FACE outcome measures were analyzed using a 1-way ANOVA with Tukey’s post-hoc tests (Supplemental Table 1). Only FACE groups with three or more pools were included in the statistical analysis.

RESULTS

Histological evaluation of Achilles tendon and ECM changes following TGFβ1-induced injury to the bursa and subsequent rHuPH20 treatment

Following injection of TGFβ1 into the RCB, Safranin-O stained sections of the ankle joint (Figure 1) showed hyperplasia of the tendon body (GAG accumulation) at the acute stage, as shown previously for intra-tendinous TGFβ1 injection injury 9. Chondroid metaplasia (a hallmark of Achilles tendinopathy) was also apparent in the sections at the acute stage, a characteristic that showed a marked reduction following +rHuPH20 application. By 9d, GAG presence in the tendon body appeared to be reduced but showed increased staining in the fat pad and surrounding tissues (anterior to the tendon). Following 25d, both +TGFβ1 and +rHuPH20 treated sections showed chondroid removal and a normal appearance of the Achilles tendon and peritenon (closely resembling that of the uninjured tendon), suggesting resolution of the injury induced cell and matrix changes.

Figure 1:

Figure 1:

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Histopathological evaluation of Achilles tendon and surrounding tissues following TGFβ1 induced injury and treatment with rHuPH20. Tissue sections were treated and stained with Safo-O described in the Methods. TGFβ1 injection resulted in cell proliferation throughout the fat pad and the peritenon (Acute and Day 9, black arrow heads), as well as swelling and chondroid metaplasia in the tendon body (Acute, white arrow heads). The rHuPH20 administration did not significantly alter histological appearance of any tissue. Note: different Fast-Green staining at 9 and 25d (+TGFβ1) was due to a different batch of dye used. C: Calcaneus, FP: Fat Pad, TB: Tendon Body, B: Retrocalcaneal Bursa.

HA Accumulation Following TGFβ1 Injection and Removal after rHuPH20 Injection

Previously, we observed that TGFβ1 injection into the tendon greatly increased HA deposition in the tendon body and surrounding tissues 6. Similarly, injection of the growth factor into the bursal region also increased HA in the acute and 9d time points relative to naïve uninjured sections, with significant clearance at the later time point (Figure 2, left hand panels). Notably, following rHuPH20 administration, there was a rapid and extensive clearance of the accumulated HA and this persisted throughout the experimental period.

Figure 2:

Figure 2:

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Efficacy of rHuPH20 injections in removing TGFβ1 induced HA accumulation during the acute phase. Tissue sections were prepared and stained for HA with bTSG6, as described in the Methods. TGFb1 injection markedly increased HA deposition in the fat pad, peritenon, and tendon body (White Arrows Acute and Day 9). rHuPH20 injection resulted in robust clearance of the HA accumulation (Black Arrows) with sustained HA clearance from the tendon body (*) throughout the experimental period. C: Calcaneus, FP: Fat Pad, TB: Tendon Body, B: Retrocalcaneal Bursa

To confirm the histological evaluation, the contents of HA in the Achilles tendons were determined biochemically using FACE analyses (Figure 3, left hand panels). Consistent with the histochemical analyses, a ~4-fold increase in HA contents was seen after acute TGFβ1 injection, which decreased by 9d to ~ 2-fold and had normalized to uninjured control levels by 25d. Following rHuPH20 injection, the post-TGFβ1 deposited HA was largely removed but then recovered to essentially uninjured naïve contents by 9d, without significant changes by 25d (Figure 4A).

Figure 3:

Figure 3:

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Abundance of HA and CS/DS in tendons from joints injected with TGFβ1 (black columns) or TGFβ1 +rHuPH20 (gray columns). Tendons were dissected, digested with proteinase K and analyzed for HA and CS/DS content using FACE as described in the Methods. Results are shown as fold changes in content per tendon relative uninjured tendons (These values were 0.050 ug HA and 0.359 ug CS/DS per tendon for 12-week old mice; 0.054 ug and 0.463 ug CS/DS per tendon for 16-week old mice. See supplemental Table 1 for number of biological replicates and number of tendons per pool analyzed. * denotes statistically significant difference compared to naïve age-matched control

Figure 4:

Figure 4:

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Localization of HA (with bTSG6, see Methods) (A) and aggrecan core proteins (with anti-DLS, see Methods) (B) in bone-tendon insertion sites (top panels) and the tendon body (bottom panels). Control sections are available in previous study by Bitterman et al. 6, Supplemental Figure 1. While rHuPH20 effectively removed HA and CS/DS from the tendon body, it had minimal effect on aggrecan core protein abundance. Horizontal bar = 50 um

FACE analyses of the proteinase K digests also provided data for the contents of CS/DS in the tendons (Figure 3, right hand panels). As expected from our previous studies, TGFβ1 caused a pronounced (~15 fold) increase in CS/DS content which returned to essentially uninjured levels. Given the known enzymatic cleavage specificity of hyaluronidase 23, 24, it can also depolymerize CS/DS. Thus, intra-bursal injection of PH20 effectively cleared these GAG chains from the tendon body/peritenon, with the contents approximating uninjured levels at 9d. Notably, TGFβ1 injected tendons had attained uninjured CS/DS levels by 25d, but in rHuPH20 treated tissues it had doubled (2.34-fold, p=0.04) over uninjured levels. No significant differences in the CS/DS sulfation ratios was observed between groups, which were ~1:10:89 for 0S:4S:6S.

To assess whether the rHuPH20 treatment had affected the aggrecan content of the tissue, sections were immunostained with the aggrecan core protein antibody, anti-DLS. As previously reported for intra-tendinous TGFβ1, intra-RCB injection also resulted in aggrecan accumulation within the tendon body (Figure 4B). Interestingly, while rHuPH20 effectively removed both HA and CS/DS from the tendon body (Figure 3A) it had minimal effect on aggrecan core protein abundance (Figure 4B).

QPCR Array Assay of Cell Fate, Energy Metabolism, ECM Remodeling and Innate Inflammation Genes in tendon and peritenon.

Gene expression assay by QPCR showed that all genes included in the array plate showed detectable levels of expression, and as expected, collagen genes were most highly expressed, whereas cytokines and chemokines showed low abundance levels of mRNA (Supplemental Figure 3). Genes also assayed in tendons at acute, 9d, and 25d post-RCB TGFβ1 injections and data are summarized in Figure 5 and Supplemental Figure 4 (+TGFβ1 panels). Only genes exhibiting statistically significant, > 2-fold changes over uninjured levels are included. A moderate response was seen at all 3 experimental time points: in the acute phase, 9 genes were increased, 2 in the cell fate group (Rel, Tlr2), 2 in the ECM remodeling group (Dcn, Has1) and 5 in the inflammation group (Cxcl1, Ccl7, Il1b and Il6), with Gapdh, Pkm and Infb1 decreased; by 9d, Homox1, Rel, Cxcl10, Mif Col3a1, Col5a1 and Mmp9 were notably increased, with the latter 3 indicative of active collagen matrix remodeling. By 25d, a reactivation of Cxcl1, Il1b and Il6 and a decrease in Cebpb, Il12a and Mmp3 had occurred. The decrease in Mmp3 indicated a resolution of the injury responses as previously reported 10.

Figure 5:

Figure 5:

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Fold change expression of select genes following TGFβ1 injection and rHuPH20 injections. Fold changes calculated relative to uninjured age-matched controls

When gene expression changes in the +TGFβ1+rHuPH20 groups (Supplemental Figure 4) were examined, a more pronounced response was seen, particularly at the acute and 9d time points. However, to establish which of the gene modifications were due to rHuPH20 injection specifically, rather than a second set of injections into the injured RCB, we performed sham PBS injections and assayed the corresponding gene responses at the acute and 9d time points (Figures 5 and 6). The data shown were calculated as fold changes relative to the TGFβ1 injection alone. We identified four distinct responses: Firstly, decreased expression of Odc1 and Slc2a1 at both time points by either PBS or rHuPH20 injection. Secondly, increased expression of Mmp3, Cxcl1, Cxcl5, Il1b, Il6 and decreased expression of Il12a only in the acute phase after either PBS or rHuPH20 injection. Thirdly, decreased expression of Col3a1, Itga5, Itgam, Serpin1, P4ha1, Pfk1, Pglyrpl was triggered by PBS only in the acute phase, and then decreased at 9d by both types of injection. Fourthly, and most significantly, 15 genes were affected specifically by rHuPH20 injection: Ier3, Rel, Tlr2, Tnfrsf1b (Cell Fate), Adora2b, Cdh1, Dcn, Has1, Wisp1 (ECM turnover), Pkm (energy metabolism), Ccl2, Ccl7, Cd80, Cxcl10, F10, Infb1, Mif, Il12a, Ptx3 (inflammation). All rHuPH20 specific responses were detected as increased expression and occurred in the acute phase, except for Ier3 which was decreased and only at 9d. In summary, the data suggest robust response of the tendon cells to the rHuPH20 injection, that largely consisted of increased activation of ECM turnover and innate inflammation responses in the acute phase, i.e. ~ 6 h after the enzyme injection.

Figure 6:

Figure 6:

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Fold change expression of select genes following rHuPH20 and PBS injections. Fold changes calculated relative to age-matched untreated injured group (TGFβ1 injections only)

Effect of TGF-β1 injection and rHuPH20 administration on tendon geometric and biomechanical properties

Statistically significant changes in maximum load were observed at the acute time point, in which +TGFβ and +rHuPH20 exhibited decreased maximum loads relative to uninjured (p<0.0001) (Figure 7). Alterations in maximum stress were observed at all three time points, but were most apparent in the acute time point, where +TGFβ and +rHuPH20 exhibited decreased maximum stress relative to uninjured (p<0.0001). These significant changes (at the acute time point) mirrored those of the stiffness (p<0.0001) and elastic modulus (p<0.001). CSA of +TGFβ at 9d was increased relative to uninjured and +rHuPH20 (p=0.0153), while maximum stress (p=0.0178) and modulus (p=0.0119) were significantly decreased. Additionally, +rHuPH20 exhibited statistically significant increases in maximum stress (p=0.0445), stiffness (p=0.0125), and modulus (p=0.0016) at the acute time point. At the 25d time point, maximum stress, stiffness, and modulus were significantly decreased in the +rHuPH20 group relative to the +TGFβ group. No significant differences were observed for viscoelastic properties.

Figure 7:

Figure 7:

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Effect of TGFβ and rHuPH20 administration on Achilles tendon mechanical properties. The scatter plots show data for individuals tendons tested in each group. For each individual group, horizontal lines denote mean +/− one standard deviation. Horizontal lines above the plots denote statistically significant differences between groups (p<0.05)

DISCUSSION

In the current study we examined a novel therapeutic approach for the dissolution of proinflammatory HA and CS/DS proteoglycan-rich ECM to aid in the healing of tendinopathy and achieve a biomechanically robust tendon, devoid of inflammation induced scarring 6. Our proposed use of a human recombinant hyaluronidase, rHuPH20, here was as an injectable treatment to degrade the acutely deposited HA matrix in response to the TGFβ1 injury 6. To assess the ability of rHuPH20 to degrade such a GAG-rich matrix in the Achilles/calcaneus complex after injection, we developed an injury model to elicit an inflammatory response by injection of TGFβ1 into the RCB. This approach eliminates the need for multiple intra-tendinous injections and furthermore enables expanded analysis of the injury response within the peri-Achilles soft tissue environment. A particular focus of this investigation was to examine the efficacy with which the enzyme could diffuse into areas of high GAG concentration, and this was most evident in the early injury response, during which rHuP20 effectively cleared the GAG-rich matrix within and surrounding the Achilles tendon.

The current results showed that, similar to our intra-tendinous TGFβ1 injury model 6, 7, 9, 10, 12, 19, TGFβ1 injection into the RCB induced rapid and pathologic formation of an inflammatory tendon matrix rich in HA and CS/DS (Figures 1–4). This is consistent with the recent finding that intra-articular injection of TGFβ1 upregulates inflammatory gene profiles and pathologic changes in peri-articular tissues34. However, our histologic analyses (Figure 1, 2) indicate a dramatic increase of sulfated GAGs, HA, and CS/DS not only in the RCB, but extending further to surrounding structures such as the tendon and fat pad. Similar to our recent intra-tendon injury model 6, clearance of the pro-inflammatory matrix (both within the tendon and peri-tendinous tissues including the RCB) occurs following TGFβ1 injection within 25 days post-injury. Gene expression results for the early post-injury response of the tendon cells also exhibited similarities to those previously published: Rel, Pkm as well as the common 9d time point: Col3a1, Col5a1, Mmp9, Mif, Cxcl5, Cxcl10; however, more subtle changes were elicited after intra-bursal injection. Furthermore, tendon biomechanical alterations accompanying this novel RCB injection model are consistent with those of our intra-tendinous injection model. In particular, at the acute and 9d time points, tendons of the TGFβ1 injected RCB group exhibited an increase in CSA, as well as decreases in maximum load, maximum stress, stiffness, and modulus relative to uninjured controls. However, unlike the intra-tendinous injury model (in the absence of mechanical stimulation) 9, at the longer evaluation time point (25d), the pathologic changes elicited by the TGFβ injection appear to resolve.

Our data suggest that rHuPH20 accelerates resolution of the TGFβ1 injury, particularly in the acute and 9d post-injury responses (Figures 1–4). The enzyme is effective in not only removing HA and CS/DS from the injection site (Figure 2), but also in the tendon body (Figures 3). Notably, although GAGs were digested, there were no significant changes in aggrecan core protein abundance (Figure 4), indicating that it is likely not immobilized on HA, but stabilized in the pericellular and fibrous ECM via other mechanisms 25. The acute depletion of HA was followed by an accumulation of newly synthesized HA to essentially uninjured naïve levels by 9d and was maintained at 25d. Similar responses were seen with the CS/DS contents, which in tendons from rHuPH20 injected animals rose (at 25d) to approximately double the content of that seen in uninjured naïve tendons (Supplemental Table 2). To what extent the rHuPH20 treatment affected contents of versican or the small leucine rich proteoglycans, decorin and biglycan remains to be examined. However, it was noted that Dcn expression was specifically upregulated by rHuPH20 in the acute phase and again at 25d (Figure 5).

The QPCR gene expression data (Figures 5 and 6) indicate that rHuPH20 has a distinct impact (vs PBS sham injection) on TGFβ1-modified genes. Such targeted increases in expression were only seen in the acute phase and included two cell fate genes (Rel and Adora2b), four ECM metabolism genes (Cadh1, Dcn, Has1, Wisp1) and seven innate inflammation genes (Ccl2, Ccl7, Cd80, F10, Mif, Ptx3 and Tlr2). Future studies are needed to clarify whether these changes are induced by altered ECM interactions in a GAG-depleted environment 12, 26 or stimulated by degradation fragments of the rHuPH20 such as HA oligosaccharides 27–32.

Our data indicate that rHuPH20 accelerates resolution of the TGFβ1 injury with regard to tendon biomechanical properties. In the acute phase, both structural and material properties of rHuPH20 treated tendons were significantly improved relative to +TGFβ1 tendons, despite the substantial reduction of tensile properties (relative to uninjured tendons) (Figure 7). TGFβ1 injury induced tendon swelling (significant increase in cross-sectional area) and a corresponding reduction (p<0.05) in material properties at the 9d time point, and both geometric and material properties following rHuPH20 treatment were restored to uninjured levels (Figure 7). In association with these indices of healing, tendon HA and CS/DS content was dramatically reduced (to levels below those of uninjured tendons) at the acute time point and hyaluronan content for the rHuPH20 group was reduced at 9d relative to the +TGFβ treated mice. Due to the resolution of these key biomechanical outcome measures, we conclude that the rHuPH20 treatment accelerated healing of Achilles tendinopathy in this murine model.

In conclusion, the current study provides supportive pre-clinical evidence of the efficacy of rHuPH20 for targeting the pro-inflammatory matrix of the Achilles tendon and adjacent retrocalcaneal bursa. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the RCB and tendon strongly supports the future refinement of clinically safe, injectable glycosidase preparations 33–35 as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse 26, 36.

Supplementary Material

Supp figS1-5&T1-2

Supplemental Table 1 (Filename: SNR_SupTable1.tif): Experimental groups and outcome measures, indicating the number of animals utilized for each group

Supplemental Figure 1 (Filename: SNR_SupFig1.tif): Experimental timeline indicating the timing of injections and euthanasia

Supplemental Figure 2 (Filename: SNR_SupFig2.tif): Gene listing of Taqman custom array plate used for QPCR assays

Supplemental Figure 3 (Filename: SNR_SupFig3.tif): DeltaCt values of naïve uninjured samples for genes sorted from highest to lowest. All DeltaCt values calculated relative to B2m housekeeping gene

Supplemental Figure 4 (Filename: SNR_SupFig4.tif): Fold change expression of select genes following TGFβ1 injection and rHuPH20 injections. Fold changes were calculated relative to uninjured age-matched controls

Supplemental Table 2 (Filename: SNR_SupTable2.tif): Abundance of HA and CS/DS in tendons from joints injected with TGFβ1 or TGFβ1+rHuPH20, reported as raw data values. Tendons were dissected, digested with Proteinase K and analyzed for HA and CS/DS content using FACE as described in the Methods

Supplemental Figure 5: (Filename: SNR_SupFig5.tif): Injection procedure for TGF-β1 and rHuPH20. a) Diagrammatic overlay onto histological image; B) Placement of 28G syringe into right hindlimb under anesthesia. C) Skin removed following injection of 10 μL India Ink to illustrate its localization to the bursa and fat pad, but not the tendon body.

ACKNOWLEDGEMENTS

This investigation was supported by NIH grant AR 63144 (VMW, AP, JL) and the Katz Rubschlager Endowment for OA Research (AP, JL). rHuPH20 and bTSG6 were provided by Halozyme Therapeutics through a MTA with Rush University.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp figS1-5&T1-2

Supplemental Table 1 (Filename: SNR_SupTable1.tif): Experimental groups and outcome measures, indicating the number of animals utilized for each group

Supplemental Figure 1 (Filename: SNR_SupFig1.tif): Experimental timeline indicating the timing of injections and euthanasia

Supplemental Figure 2 (Filename: SNR_SupFig2.tif): Gene listing of Taqman custom array plate used for QPCR assays

Supplemental Figure 3 (Filename: SNR_SupFig3.tif): DeltaCt values of naïve uninjured samples for genes sorted from highest to lowest. All DeltaCt values calculated relative to B2m housekeeping gene

Supplemental Figure 4 (Filename: SNR_SupFig4.tif): Fold change expression of select genes following TGFβ1 injection and rHuPH20 injections. Fold changes were calculated relative to uninjured age-matched controls

Supplemental Table 2 (Filename: SNR_SupTable2.tif): Abundance of HA and CS/DS in tendons from joints injected with TGFβ1 or TGFβ1+rHuPH20, reported as raw data values. Tendons were dissected, digested with Proteinase K and analyzed for HA and CS/DS content using FACE as described in the Methods

Supplemental Figure 5: (Filename: SNR_SupFig5.tif): Injection procedure for TGF-β1 and rHuPH20. a) Diagrammatic overlay onto histological image; B) Placement of 28G syringe into right hindlimb under anesthesia. C) Skin removed following injection of 10 μL India Ink to illustrate its localization to the bursa and fat pad, but not the tendon body.

NIHMS1048838-supplement-Supp_figS1-5_T1-2.pdf (734.2KB, pdf)

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