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The Scope of Hyaluronic Acid as an Experimental Intraocular Implant
The Scope of Hyaluronic Acid as an Experimental Intraocular Implant ERWIN HULTSCH, MD, PhD
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Abstract: The inflammatory potential of hyaluronic acid (HA) varies from batch to batch of HA independent of tissue origin, concentration, or amino acid composition. The inflammatory potential can be determined by intravitreous testing only, not by intracameral testing. Retention of HA in the eye depends on molecular weight and concentration of HA, not on the ocular inflammatory response. Concentration, molecular weight, and inflammatory grade of any HA batch used in intraocular surgery should be specified. Monitoring of the postoperative intraocular pressure is recommended. [Key words: hyaluronic acid, intraocular implant.] Ophthalmology 87:706-712, 1980
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Ever since Deutschmann in 1895 used rabbit vitreous successfully as an implant in a desperate case of human retinal detachment, 1 there has been a search for the ideal vitreous implant. Attributes of major importance are biological inertness and physicochemical properties that do not interfere with the normal transmission of light and that secure the position of a surgically reattached retina. In practice, however, it appears difficult to fulfill all desired criteria simultaneously, as demonstrated by the great number of materials that have been tested and discarded. 2 The mechanical concept of an internal plomb is the rationale for the use of hyaluronic acid (HA), a biopolymer that is also a natural comFrom the Department of Retina Research, Eye Research Institute of Retina Foundation, and Department of Ophthalmology, Harvard Medical School, Boston.
ponent of the vitreous. At physiologic pH, HA is a viscoelastic fluid (not a gel), thus providing a nonpermanent implant material. Previous well-documented animal studies have reported considerable inflammatory reactions after intravitreous injection of HA, especially in cases of altered vascular permeability. a-s These reactions consisted of flare, vitreous haze, and great numbers of inflammatory cells. In the course of our investigations of experimental vitreous implants, we obtained HA preparations that were well tolerated. A quantitative evaluation of the inflammatory response to and comparative analysis of such preparations has not yet been reported. The purpose of this experimental study was therefore to test and quantitate the inflammatory response to these preparations as well as the outflow of HA following injection into the vitreous and the anterior chamber of the owl monkey eye.
Presented at the Eighty-Fourth Annual Meeting of the American Academy of Ophthalmology, San Francisco, November 5-9, 1979.
MATERIALS AND METHODS
Supported in part by US-PHS grants EY-02895, EY -02427, and the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to Erwin Hultsch, MD, Eye Research Institute of Retina Foundation, 20 Staniford Street, Boston, MA 02114.
706
INTRAOCULAR IMPLANTS
All injected solutions were sterile. The hyaluronic acid solutions were prepared from bovine vitreous (BV; Etamucine, Chibret
0161-6420/80/0700/0706/$00.85 © American Academy of Ophthalmology
Laboratories, Clermont-Ferrand, France), human umbilical cord (HUC; Biotrics, Inc., Arlington, Mass.), and rooster comb (RC; Biotrics, Inc., Arlington, Mass.; Med.-Chem. Products Inc., Waltham, Mass.; Pharmacia, Uppsala, Sweden). The HUC and RC preparations had an HA concentration of 10.0 to 10.6 mg/ml (solvent 0.15 M phosphate buffer, pH 7.2 or 0.15 M NaCl) and an intrinsic viscosity of 2200 to 2600 ml/g unless otherwise indicated. BV- HA had a concentration of 2 mg/ml (pH 7.2, solvent not specified) and an intrinsic viscosity of 1100 ml/g. Physiologic saline (0.15 M) served as control.
CHEMICAL ANALYSES
Vitreous and aqueous samples were diluted and then centrifuged for one hour at 40,000 x g; the supernatant was then extensively dialyzed against 0.15 M NaCl at 4°C. Vitreous and aqueous protein content was determined with the Lowry method. Hyaluronic acid concentration was calculated from the hexuronic acid content, using a modified 6 carbazole reaction method. Viscosity measurements were carried out at 25°C in a Cannon- Ubbelohde semi-microdilution viscometer. Amino acid composition was determined as previously described. 7
PREPARATION OF ANIMALS
RESULTS
Healthy adult owl monkeys (Aotus tnvlrgatus) were used. Animals with pre-existing ocular abnormalities or with trauma caused by the operation were excluded from the study. General anesthesia was achieved by intramuscular injection of sodium pentobarbital (3 to 4 mg/100 g body weight). The pupil was dilated by two drops of 1% cyclopentolate preoperatively and two drops of 1% atropine sulfate postoperatively.
Table 1 defines our grading of the ocular inflammatory response. We extended the conventional scale of four grades of inflammation to six grades to obtain a more subtle classification of the changes. This scale also corresponded well with our laboratory findings regarding protein exudation and number of inflammatory cells in vitreous and aqueous humor. Different batches of HUC and RC hyaluronic acid preparations can cause inflammatory reactions ranging from a clinical grade + 1 to a grade +6 response at the 48-hour peak. The inflammatory reaction varies from batch to batch and can be determined by intravitreous testing only. Figure 1 compares the ocular response following intravitreous injection of saline, selected grade +2 inflammatory HA preparations (BV, HUC, RC), and selected grade +5-6 inflammatory HA solutions (HUC, RC). The inflammatory changes followed a typical pattern, with a maximum clinical response after two days, regardless of the nature of the injected material. The time course was most evident after injection of highly inflammatory HA preparations: at two days, a grade +5-6 reaction, consisting of severe aqueous and vitreous flare and great masses of inflammatory cells, was observed. The large white keratic precipitate and the nature of the fundus reflex allow a reliable classification of the reaction. At five days, the size of the keratic precipitate diminished markedly and fundus details became visible. The evaluation at two days is therefore an important parameter and is used to classify the inflammatory potential of different HA preparations and batches on a grade + 1 to grade +6 scale. The reaction to vitreous replacement with grade + 2 HA was ophthalmologically very similar to that observed after saline injection. However, as shown in Figure 1, differences can be observed during the short-term follow-up: Although the estimated number of cells in vitre-
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SURGICAL TECHNIQUE AND EVALUATION OF INFLAMMATORY RESPONSE
Under sterile conditions, a lateral canthotomy was performed, the temporal bone wall of the orbit was removed, the Tenon's capsule was opened, and the sclera was exposed. After light surface diathermy, a short (3/8 in.) 25gauge needle connected to a flexible tube (Butterfly pediatric infusion set, Abbott Laboratory, Chicago) was inserted 5 mm behind the limbus through the pars plana ciliaris into the central part of the vitreous. Fifty per cent of the owl monkey vitreous (1.1 ml) was exchanged with an equal volume of HA or saline. A preplaced mattress suture prevented leakage. The intraocular pressure remained normal (15 torr) throughout the postoperative observation period. Aqueous humor (0.3 ml) was withdrawn for analysis or replacement via a 25- to 30-gauge needle attached to a 1-ml syringe; it was replaced with air, HA, or saline solution. Inflammatory cells from the vitreous and aqueous were counted in a hemocytometer chamber. Intraocular pressure was measured with a Schi~tz tonometer without correction for the difference in the corneal diameter between human and owl monkey eyes. Eyes were examined by ophthalmoscopy, slit lamp, and biomicroscopy using a Goldmann three-mirror lens fitted to the owl monkey eye.
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Table 1. Grading of Inflammatory Response After Intravitreous HA Injection
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Aqueous and Vitreous
Clinical Grade
Flare
Cells
Cells/mm 3
None
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2
Barely detectable Slight
3
Moderate
4
Moderatesevere
5
Severe
Masses
600-1200
6
Severe
Masses
>1200
Very few
<50
Moderate number Many
50-200 200-600
ous and in aqueous humor was similar at two days, the flare in the vitreous was still detectable at 10 days following grade + 2 HA injection; eyes injected with saline were free of any inflammatory signs. All reactions are transient and disappeared completely after 10 days (saline), 20 days (grade +2 HA) and 30 days (grade +5-6 HA). The normal immediate response within 30 minutes to two hours after all modes of injection consisted of slight flare only. The long-term follow-up for up to two years after primary injection shows that all HApreparations are equally well tolerated by the owl monkey eye: Neither cells nor flare were detectable in vitreous and aqueous humor; retina, ciliary body region, lens, iris, and cornea were free of pathologic changes when examined
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Vitreous Haze and Ocular Fundus No haze Fundus clear No haze Fundus clear Definite haze Fundus clear Marked haze Fundus details just visible Severe haze Red fundus reflex, no fundus details visible Severe haze Gray fundus reflex
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None None Just detectable Small
Medium Large
ophthalmologically and under the dissecting microscope; and all monkeys appeared to have normal vision as judged by daily handling of the animals. It should be emphasized that depending on the particular HA batch, human umbilical cord as well as different rooster comb preparations (Biotrics, Pharmacia, Med.-Chem.) can produce grade +5 and +6 reactions (Fig 1) similar to that observed after injection of HA V (rooster comb HA). The latter was selected for comparative purposes. The inflammatory 48-hour response to selected grade + 2 HA preparations of different tissue origin was quantitated by analysis of protein exudation and the number of inflammatory cells in vitreous and aqueous. The reac-
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Fig 1. Clinical evaluation and follow-up of ocular inflammatory response on a + 1- +6 grade inflammatory scale (Table 1) following primary vitreous injection of: e-e, HA from human umbilical cord (HUC) and rooster comb (RC); 0 - 0 , HA from bovine vitreous (BY), HUC and RC; x----x, saline as control. Low-grade reactive HA preparations ( 0 - 0 ) and high-grade preparations HA reactive (e-e) were selected arbitrarily for comparison of the inflammatory response. All reactions were transient with a maximum at two days. (n) number of eyes.
tion was compared with that found after intravitreous injection of saline and highly inflammatory (grade +5-6) HA. The following HA preparations were studied: HA I, bovine vitreous HA (Etamucine, Chibret); HA II, human umbilical cord HA (Healon-H, Biotrics); HA III, rooster comb HA (Pharmacia); HA IV and V, rooster comb HA (Highvisc, Med.Chem.). Table 2 shows that preparations HA I- IV caused a mild inflammatory response comparable to that observed after saline injection regardless of their tissue origin, HA concentration, or preparation methods used. The average 48-hour concentration of exudative proteins (0.5 ± 0.3 mg/ml vitreous) increases seven times over that of the original vitreous (0.07 ± 0.03 mg/ml vitreous). Vitreous and aqueous cell counts and protein values show no statistically significant difference when HA I- IV preparations are compared with the control injection of saline. The cause of the inflammatory reaction to grade +5-6 HA is not known. It is not correlated to the tissue origin (Table 2), the molecular weight of HA, the total protein content, or the amino acid composition of the residual protein associated with the different HA preparations (Table 3). Similarly, there is no difference in the retention time of grade + 2 and grade +5-6 HA preparations in the vitreous (Table 3). Injection of HA into the anterior chamber of the owl monkey eye does not allow differentiation between grade +2 and grade +5-6 inflammatory HA as demonstrated by Table 4. Both preparations caused a grade + 2 response in the anterior segment at two days. Keratic precipitates were not seen. The posterior segment was not affected by the anterior chamber injection and remained quiet without any inflammatory
changes. The average concentration of exudative proteins is the same (5.2 mg/ml aqueous) when grade +2 and grade +5-6 HA preparations are compared. However, both reactions are moderately increased over the cellular response observed after anterior chamber injection of saline. Saline injection resulted in 5 cells/mm3 and 3.5 mg protein/ml aqueous at two days, which in turn was elevated over the original protein concentration of the aqueous (0.28 mg/ml) as determined by paracentesis. There is no significant difference in the outflow of grade + 2 and grade +5-6 HA from the anterior chamber; 5% and 7% of the injected material is retained in the aqueous humor 24 hours after injection. 1-
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The present investigation demonstrates that the inflammatory potential of HA varies from batch to batch of tested HA. Thus, following vitreous injection, HA may cause only a slight inflammatory response or result in inflammatory changes corresponding up to grade +5-6 reactions on a grade + 1.-- + 6 scale. The slight + 2 inflammatory response is almost indistinguishable ophthalmologically from that observed after injection of saline solution. This is in contrast to previous studies reporting considerable inflammatory reaction in animal eyes following injection of HA prepared from bovine vitreous, human umbilical cord, or rooster comb. 3 - 5 •8 •9 Quantitation of the inflammatory changes can be achieved by cell counts and chemical analysis of exudative proteins, provided that the specimen can be withdrawn without undue
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Table 2. Protein Concentration and Cell Count in Aqueous and Vitreous 2 Days After Primary Vitreous Replacement Aqueous
Vitreous
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Injected Material Concentration of HA
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Saline HA I (BV) 2 mg/ml HA II (HUC) 10 mg/ml HA II (HUC) 18 mg/ml HA Ill (RC) 10 mg/ml HA IV (RC) 10 mg/ml HA V (RC) 10 mg/ml
N
Mean Protein Concentration mg/ml
4
5.7 (± 5.7)
7 (± 4)
4
0.25 (± 0.16)
13 (± 23)
2
2
4.4(±1.1)
29 (± 13)
2
0.29 (± 0.09)
1 (± 2)
2
6
7.3 (± 5.6)
5 (± 5)
4
0.26 (± 0.03)
1 (± 2)
2
2
8.6 (± 4.6)
8 (± 7)
2
0.31 (± 0.05)
0
2
4
6.3 (± 4.8)
40 (± 28)
4
1.06 (± 0.27)
42 (± 32)
2
4
2.0 (± 0.9)
5 (± 7)
4
0.58 (± 0.12)
6 (± 10)
2
4
34.8 (± 11.9)
1360 (± 1290)
4
4.6(±1.1)
Mean No. of Cells/mm 3
N
Mean Protein Concentration mg/ml
Mean No. of Cells/mm 3
----
1824 (± 976)
Clinical Grade -------
5-6
709
Table 3. Chemical Properties of Tested HA Preparations Preparation (Tissue Origin) 48-Hr Inflammatory Grade Concentration Intrinsic Viscosity HUG +2 10 mg/ml 2400 ml!g RC* +2 10 mg/ml 2200 ml/g RC +2 11 mg/ml 4400 ml/g RCt +5-6 10 mg/ml 2400 ml!g
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Amino Acid Composition ~-tg/mg HA Ala Gly Glu Ser
lieu
Leu
Asp
Thr
0.64
0.11
0.07
0.08
0.16
0.06
0.07
0.02
0.08
0.75
0.11
0.06
0.10
0.18
0.11
0.09
0.03
0.07
0.27
0.04
0.02
0.02
0.05
0.04
0.03
0.03
0.04
0.29
0.07
0.02
0.03
0.07
0.04
0.02
0.02
0.02
92% of injected HA was retained in vitreous at 48 hr. of injected HA was retained in vitreous at 48 hr.
t 94%
mechanical trauma. The owl monkey vitreous provides a very sensitive and reproducible biological test system because more than 90% of its vitreous volume is a collagen-freeH1 viscous liquid and not a rigid gel like that of other primates (except the bush baby) or the rabbit. Thus, the vitreous can be withdrawn and replaced with little or no mechanical damage, and the variation of the response to the same injected material is kept at a minimum. Clinical evaluation in combination with vitre- · ous and aqueous humor cell counts and Lowry protein determinations is necessary because of the high standard deviation of the protein exudation into the vitreous (average standard deviation ±22%) and especially the aqueous humor (average standard deviation ±52%). The combination of clinical and laboratory findings,
however, allows a reliable evaluation of the inflammatory potential of the particular HA batch on a grade + 1 to +6 scale provided that the preparation is· tested by injection into the vitreous. The HA preparations resulting in greater than a grade +2 reaction (corresponding to more than 50 cells/mm3 of vitreous and aqueous and an average of0.5 mg Lowry protein/ml vitreous and 5.7 mg Lowry protein/ml aqueous) should preferably not be used in human vitreous surgery. It must be emphasized, however, that even the severe inflammatory changes due to highly inflammatory (grade +5-6) HA injection are transient and do not cause late or lasting pathologic changes in the normal owl monkey eye up to two years after primary injection. The slight inflammation following injection of grade
Table 4. Aqueous Inflammatory Response to Primary Anterior Chamber Injection of Saline and HA
Treatment
N
Replaced Volume of Aqueous ml
Paracentesis Saline HA-(HUC) HA-(RC)
16 6 6 4
0.3 0.3 0.3 0.3
Hours after Injection 0 48 48* 48t
N = number of eyes. * 5% of injected HA was retained in aqueous humor at 24 hr. t 7% of injected HA was retained in aqueous humor at 24 hr. :j: 48-hr inflammatory reaction after intravitreous injection.
710
Mean Protein Concentration mg/ml 0.28 3.5 5.2 5.2
(± (± (± (±
0.21) 4.3) 1.9) 6.1)
Mean No. of Cells/mm 3
0
5 (± 5) 26 (± 14) 59 (± 36)
Clinical Grade in Anterior Segment 2 (2:j:) 2 (2:j:) 2 (5-6:j:)
+2 inflammatory HA, on the other hand, is acceptable for ocular surgery because it is comparable to the ocular response to saline. Injection of HA into the anterior chamber cannot serve as a test system for differentiation between grade + 2 and highly inflammatory HA because our data show that both preparations are almost equally well tolerated in the anterior segment. Therefore, this more convenient approach cannot substitute for intravitreous testing. The consequence, however, is that grade +5-6 inflammatory HA may be as safely used as grade + 2 inflammatory HA in surgery on the anterior segment (eg, implantation of intraocular lenses). Furthermore, HA injected into the anterior chamber does not diffuse into the vitreous, provided that the anterior hyaloid membrane is intact (Hultsch, unpublished data). The cause of the severe inflammation following intravitreous HA injection is not known and deserves further study; it is not caused by contamination with endotoxins. It appears that five minutes of autoclaving at l20°C reduces the inflammatory potential from a grade +5-6 to a grade +3 reaction. Unfortunately, this approach is not practical because it is accompanied by a significant depolymerization of HA (eg, from an intrinsic viscosity value of 2000 mVg to a value of 300 mVg). Exposure of the posterior segment (ciliary body?) is apparently a prerequisite for the development of inflammatory sequelae. As the present communication demonstrates, tissue origin, protein content, and amino acid composition are not correlated to the inflammatory potential. Thus, HA extracted from bovine vitreous, the rooster comb, and human umbilical cord may be equally well tolerated. Similarly, the inflammatory response to HA appears not to be correlated to the concentration used, provided that the preparation is of a low reactive grade. Thus, injection of an 18 rng/ml solution resulted in an inflammatory response comparable to that observed after injection of the lower concentrated (10 mg HA/ml) solution of the same umbilical cord batch. Regarding the inflammatory potential of different HA batches, preparations obtained from Med.-Chem. Products exhibited the highest variability, whereas those obtained from Pharmacia showed the lowest variability in inflammatory response. The chemical and inflammatory properties of HA preparations, packaged in glass syringes sealed in air-tight plastic bags, were unaltered over a period of five years. The higher molecular weight HA obtained from umbilical cord and rooster comb is more likely to be retained for longer periods of time in the human eye than the lower molecular weight bovine vitreous HA because the outflow of HA
from the vitreous depends on the molecular weight of the injected biopolymer. 11 Preliminary data obtained from post-open sky vitrectomy specimen (Hultsch and Schepens, unpublished data) indicate that in the human eye, injected HA is retained for significantly longer periods of time (up to 20 days) than in the owl monkey eye, which may explain the beneficial effect observed after the clinical use of low molecular weight HA (Etamucine)P A potential danger might be the development of glaucoma in predisposed human eyes; close monitoring of the intraocular pressure is therefore advisable. It is important to note that intraocular inflammation neither degrades HA nor results in an accelerated outflow from vitreous and aqueous. Similarly, experimental argon and krypton laser irradiation does not alter the structure of vitreous HA . 13 That is, laser coagulation therapy following intraocular HA injection will not diminish the tamponading effect of HA. The following characteristics of HA are of special importance for its use as intraocular implants and should be known to the ocular surgeon: (1) Concentration and molecular weight, both of which determine the rate of outflow from the eye via the anterior hyaloid membrane and the Schlemm's canal. Hyaluronic acid concentrations of 10-20 mg HA/ml solvent and intrinsic viscosity values of 2000-4000 mVg (preferably more than 3000 ml/g) will result in a long-lasting tamponading effect. (2) The inflammatory potential, which varies from HA batch to HA batch; it can be determined and quantitated by intravitreous testing only. The 48-hour inflammatory response should be tested in four eyes (preferably owl monkey eyes) and should not exceed the response to control injection of salinecorresponding to a grade + 1 - + 2inflammatory reaction on a grade + 1- +6 scale with less than 50 inflammatory cells/ml vitreous and aqueous, less than 0.5 mg Lowry protein/ml vitreous, and less than 6 mg Lowry protein/ml aqueous humor. Hyaluronic acid forms a gel only at a low pH (pH 2.5); the gel is therefore not usable for intraocular surgery. The viscoelastic fluidity of Na-hyaluronate at physiologic pH, on the other hand, makes the biopolymer easily injectable and provides a nonpermanent implant material suitable for repeated use. Our data on mechanisms controlling the macromolecular composition and regeneration of the vitreous in animal models14•15 provide a basis for the analysis of human material. Our experimental finding that HA reduces the influx of inflammatory proteins into the vitreous 11 indicates a regulatory function of HA; this may
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prove to be of additional importance for the use of HA in intraocular surgery. Of further importance for the retention of HA in the intraocular fluids is the presence of a novel factor, HADPF, in vitreous and aqueous humor. 16 HADPF (Hyaluronic Acid-Depolymerization Prevention Factor) inhibits the ascorbic acid-induced degradation of HA, thus reducing the outflow of injected HA.
ACKNOWLEDGMENT
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The assistance of J. W. Appleby and B. Pietrasz and the critical review of the manuscript by Dr. Alice Adler are gratefully acknowledged.
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REFERENCES 1. Deutschmann R. Ueber ein neues Heilverfahren bei Netzhautabl~sung. Beitr Augenheilkd 1895; 20:1-80. 2. Peyman GA, Ericson ES, May DR. A review of substances and techniques of vitreous replacement. Surv Ophthalmol 1972; 17:41-51 . 3. Constable IJ, Swann DS. Biological vitreous substitutes. Inflammatory response in normal and altered animal eyes. Arch Ophthalmol 1972; 88:544-8. 4. Widder W. Hyalurons aure als GlaskDrperimplantat bei Netzhautablosung. Albrecht von Graefes Arch Klin Exp Ophthalmol 1960; 162:416-29. 5. Kl~ti R. Die heutigen Probleme in der Amotio-chirurgie. Ophthalmologica 1968; 156:415-24.
6. Bitter T, Muir HM. A modified uronic acid carbazole reaction. Anal Biochem 1962; 4:330-4. 7. Peczon BD, Antreassian R, Bucay P. Chromatographic analysis of hydroxylysine glycosides and acid hydrolysates. J Chromatogr 1979; 169:351 -6. 8. Balazs EA, Sweeney DB. The injection of hyaluronic acid and reconstituted vitreous into the vitreous cavity. In: McPherson A (ed.). New and Controversial Aspects of Retinal Detachment. New York: Hoeber Medical Division, Harper & Row, 1968; 371-6. 9. KIDti R. Hyaluronsaure als GlaskDrpersubstituent. Ophthalmologica 1972; 165:351-9. 10. Chakrabarti B, Hu ltsch E. Owl monkey vitreous: a novel model for hyaluronic acid structural studies Biochem Biophys Res Commun 1976; 71:1189-93. 11. Hultsch E. Vitreous structure and ocular inflammation. In Silverstein AM, O'Connor GR (eds.). Immunology and Immunopathology of the Eye. New York: Masson, 1979; 97-103. 12. Edmund J. Vitreous substitute in the treatment of retinal detachment. Mod Probl Ophthalmol 1974; 12:370-7. 13. Hultsch E, Ducrey N. Effect of argon and krypton laser irradiation on the normal owl monkey vitreous. Mod Probl Ophthalmol1979; 20:184-7. 14. Hultsch E. Regeneration of hyaluronic acid in the owl monkey vitreous: evidence for control mechanisms. ARVO 1978. Invest Ophthalmol Vis Sci April, 1978; Suppl: 209. 15. Pietrasz B, Hultsch E. Pathophysiology of vitreous hyaluronic Acid. ARVO 1980. Invest Ophthalmol Vis Sci April, 1980; Suppl: 232. 16. Hultsch E, Mukherjee DC. Studies on the comparative deopylmerization behavior of native and purified hyaluronic acid by ascorbic acid oxidation. Fed Proc 1980; 39:1637.
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Abstract: The inflammatory potential of hyaluronic acid (HA) varies from batch to batch of HA independent of tissue origin, concentration, or amino acid composition. The inflammatory potential can be determined by intravitreous testing only, not by intracameral testing. Retention of HA in the eye depends on molecular weight and concentration of HA, not on the ocular inflammatory response. Concentration, molecular weight, and inflammatory grade of any HA batch used in intraocular surgery should be specified. Monitoring of the postoperative intraocular pressure is recommended. [Key words: hyaluronic acid, intraocular implant.] Ophthalmology 87:706-712, 1980
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Ever since Deutschmann in 1895 used rabbit vitreous successfully as an implant in a desperate case of human retinal detachment, 1 there has been a search for the ideal vitreous implant. Attributes of major importance are biological inertness and physicochemical properties that do not interfere with the normal transmission of light and that secure the position of a surgically reattached retina. In practice, however, it appears difficult to fulfill all desired criteria simultaneously, as demonstrated by the great number of materials that have been tested and discarded. 2 The mechanical concept of an internal plomb is the rationale for the use of hyaluronic acid (HA), a biopolymer that is also a natural comFrom the Department of Retina Research, Eye Research Institute of Retina Foundation, and Department of Ophthalmology, Harvard Medical School, Boston.
ponent of the vitreous. At physiologic pH, HA is a viscoelastic fluid (not a gel), thus providing a nonpermanent implant material. Previous well-documented animal studies have reported considerable inflammatory reactions after intravitreous injection of HA, especially in cases of altered vascular permeability. a-s These reactions consisted of flare, vitreous haze, and great numbers of inflammatory cells. In the course of our investigations of experimental vitreous implants, we obtained HA preparations that were well tolerated. A quantitative evaluation of the inflammatory response to and comparative analysis of such preparations has not yet been reported. The purpose of this experimental study was therefore to test and quantitate the inflammatory response to these preparations as well as the outflow of HA following injection into the vitreous and the anterior chamber of the owl monkey eye.
Presented at the Eighty-Fourth Annual Meeting of the American Academy of Ophthalmology, San Francisco, November 5-9, 1979.
MATERIALS AND METHODS
Supported in part by US-PHS grants EY-02895, EY -02427, and the Massachusetts Lions Eye Research Fund, Inc. Reprint requests to Erwin Hultsch, MD, Eye Research Institute of Retina Foundation, 20 Staniford Street, Boston, MA 02114.
706
INTRAOCULAR IMPLANTS
All injected solutions were sterile. The hyaluronic acid solutions were prepared from bovine vitreous (BV; Etamucine, Chibret
0161-6420/80/0700/0706/$00.85 © American Academy of Ophthalmology
Laboratories, Clermont-Ferrand, France), human umbilical cord (HUC; Biotrics, Inc., Arlington, Mass.), and rooster comb (RC; Biotrics, Inc., Arlington, Mass.; Med.-Chem. Products Inc., Waltham, Mass.; Pharmacia, Uppsala, Sweden). The HUC and RC preparations had an HA concentration of 10.0 to 10.6 mg/ml (solvent 0.15 M phosphate buffer, pH 7.2 or 0.15 M NaCl) and an intrinsic viscosity of 2200 to 2600 ml/g unless otherwise indicated. BV- HA had a concentration of 2 mg/ml (pH 7.2, solvent not specified) and an intrinsic viscosity of 1100 ml/g. Physiologic saline (0.15 M) served as control.
CHEMICAL ANALYSES
Vitreous and aqueous samples were diluted and then centrifuged for one hour at 40,000 x g; the supernatant was then extensively dialyzed against 0.15 M NaCl at 4°C. Vitreous and aqueous protein content was determined with the Lowry method. Hyaluronic acid concentration was calculated from the hexuronic acid content, using a modified 6 carbazole reaction method. Viscosity measurements were carried out at 25°C in a Cannon- Ubbelohde semi-microdilution viscometer. Amino acid composition was determined as previously described. 7
PREPARATION OF ANIMALS
RESULTS
Healthy adult owl monkeys (Aotus tnvlrgatus) were used. Animals with pre-existing ocular abnormalities or with trauma caused by the operation were excluded from the study. General anesthesia was achieved by intramuscular injection of sodium pentobarbital (3 to 4 mg/100 g body weight). The pupil was dilated by two drops of 1% cyclopentolate preoperatively and two drops of 1% atropine sulfate postoperatively.
Table 1 defines our grading of the ocular inflammatory response. We extended the conventional scale of four grades of inflammation to six grades to obtain a more subtle classification of the changes. This scale also corresponded well with our laboratory findings regarding protein exudation and number of inflammatory cells in vitreous and aqueous humor. Different batches of HUC and RC hyaluronic acid preparations can cause inflammatory reactions ranging from a clinical grade + 1 to a grade +6 response at the 48-hour peak. The inflammatory reaction varies from batch to batch and can be determined by intravitreous testing only. Figure 1 compares the ocular response following intravitreous injection of saline, selected grade +2 inflammatory HA preparations (BV, HUC, RC), and selected grade +5-6 inflammatory HA solutions (HUC, RC). The inflammatory changes followed a typical pattern, with a maximum clinical response after two days, regardless of the nature of the injected material. The time course was most evident after injection of highly inflammatory HA preparations: at two days, a grade +5-6 reaction, consisting of severe aqueous and vitreous flare and great masses of inflammatory cells, was observed. The large white keratic precipitate and the nature of the fundus reflex allow a reliable classification of the reaction. At five days, the size of the keratic precipitate diminished markedly and fundus details became visible. The evaluation at two days is therefore an important parameter and is used to classify the inflammatory potential of different HA preparations and batches on a grade + 1 to grade +6 scale. The reaction to vitreous replacement with grade + 2 HA was ophthalmologically very similar to that observed after saline injection. However, as shown in Figure 1, differences can be observed during the short-term follow-up: Although the estimated number of cells in vitre-
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SURGICAL TECHNIQUE AND EVALUATION OF INFLAMMATORY RESPONSE
Under sterile conditions, a lateral canthotomy was performed, the temporal bone wall of the orbit was removed, the Tenon's capsule was opened, and the sclera was exposed. After light surface diathermy, a short (3/8 in.) 25gauge needle connected to a flexible tube (Butterfly pediatric infusion set, Abbott Laboratory, Chicago) was inserted 5 mm behind the limbus through the pars plana ciliaris into the central part of the vitreous. Fifty per cent of the owl monkey vitreous (1.1 ml) was exchanged with an equal volume of HA or saline. A preplaced mattress suture prevented leakage. The intraocular pressure remained normal (15 torr) throughout the postoperative observation period. Aqueous humor (0.3 ml) was withdrawn for analysis or replacement via a 25- to 30-gauge needle attached to a 1-ml syringe; it was replaced with air, HA, or saline solution. Inflammatory cells from the vitreous and aqueous were counted in a hemocytometer chamber. Intraocular pressure was measured with a Schi~tz tonometer without correction for the difference in the corneal diameter between human and owl monkey eyes. Eyes were examined by ophthalmoscopy, slit lamp, and biomicroscopy using a Goldmann three-mirror lens fitted to the owl monkey eye.
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Table 1. Grading of Inflammatory Response After Intravitreous HA Injection
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Clinical Grade
Flare
Cells
Cells/mm 3
None
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Barely detectable Slight
3
Moderate
4
Moderatesevere
5
Severe
Masses
600-1200
6
Severe
Masses
>1200
Very few
<50
Moderate number Many
50-200 200-600
ous and in aqueous humor was similar at two days, the flare in the vitreous was still detectable at 10 days following grade + 2 HA injection; eyes injected with saline were free of any inflammatory signs. All reactions are transient and disappeared completely after 10 days (saline), 20 days (grade +2 HA) and 30 days (grade +5-6 HA). The normal immediate response within 30 minutes to two hours after all modes of injection consisted of slight flare only. The long-term follow-up for up to two years after primary injection shows that all HApreparations are equally well tolerated by the owl monkey eye: Neither cells nor flare were detectable in vitreous and aqueous humor; retina, ciliary body region, lens, iris, and cornea were free of pathologic changes when examined
6
Vitreous Haze and Ocular Fundus No haze Fundus clear No haze Fundus clear Definite haze Fundus clear Marked haze Fundus details just visible Severe haze Red fundus reflex, no fundus details visible Severe haze Gray fundus reflex
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ophthalmologically and under the dissecting microscope; and all monkeys appeared to have normal vision as judged by daily handling of the animals. It should be emphasized that depending on the particular HA batch, human umbilical cord as well as different rooster comb preparations (Biotrics, Pharmacia, Med.-Chem.) can produce grade +5 and +6 reactions (Fig 1) similar to that observed after injection of HA V (rooster comb HA). The latter was selected for comparative purposes. The inflammatory 48-hour response to selected grade + 2 HA preparations of different tissue origin was quantitated by analysis of protein exudation and the number of inflammatory cells in vitreous and aqueous. The reac-
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Fig 1. Clinical evaluation and follow-up of ocular inflammatory response on a + 1- +6 grade inflammatory scale (Table 1) following primary vitreous injection of: e-e, HA from human umbilical cord (HUC) and rooster comb (RC); 0 - 0 , HA from bovine vitreous (BY), HUC and RC; x----x, saline as control. Low-grade reactive HA preparations ( 0 - 0 ) and high-grade preparations HA reactive (e-e) were selected arbitrarily for comparison of the inflammatory response. All reactions were transient with a maximum at two days. (n) number of eyes.
tion was compared with that found after intravitreous injection of saline and highly inflammatory (grade +5-6) HA. The following HA preparations were studied: HA I, bovine vitreous HA (Etamucine, Chibret); HA II, human umbilical cord HA (Healon-H, Biotrics); HA III, rooster comb HA (Pharmacia); HA IV and V, rooster comb HA (Highvisc, Med.Chem.). Table 2 shows that preparations HA I- IV caused a mild inflammatory response comparable to that observed after saline injection regardless of their tissue origin, HA concentration, or preparation methods used. The average 48-hour concentration of exudative proteins (0.5 ± 0.3 mg/ml vitreous) increases seven times over that of the original vitreous (0.07 ± 0.03 mg/ml vitreous). Vitreous and aqueous cell counts and protein values show no statistically significant difference when HA I- IV preparations are compared with the control injection of saline. The cause of the inflammatory reaction to grade +5-6 HA is not known. It is not correlated to the tissue origin (Table 2), the molecular weight of HA, the total protein content, or the amino acid composition of the residual protein associated with the different HA preparations (Table 3). Similarly, there is no difference in the retention time of grade + 2 and grade +5-6 HA preparations in the vitreous (Table 3). Injection of HA into the anterior chamber of the owl monkey eye does not allow differentiation between grade +2 and grade +5-6 inflammatory HA as demonstrated by Table 4. Both preparations caused a grade + 2 response in the anterior segment at two days. Keratic precipitates were not seen. The posterior segment was not affected by the anterior chamber injection and remained quiet without any inflammatory
changes. The average concentration of exudative proteins is the same (5.2 mg/ml aqueous) when grade +2 and grade +5-6 HA preparations are compared. However, both reactions are moderately increased over the cellular response observed after anterior chamber injection of saline. Saline injection resulted in 5 cells/mm3 and 3.5 mg protein/ml aqueous at two days, which in turn was elevated over the original protein concentration of the aqueous (0.28 mg/ml) as determined by paracentesis. There is no significant difference in the outflow of grade + 2 and grade +5-6 HA from the anterior chamber; 5% and 7% of the injected material is retained in the aqueous humor 24 hours after injection. 1-
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The present investigation demonstrates that the inflammatory potential of HA varies from batch to batch of tested HA. Thus, following vitreous injection, HA may cause only a slight inflammatory response or result in inflammatory changes corresponding up to grade +5-6 reactions on a grade + 1.-- + 6 scale. The slight + 2 inflammatory response is almost indistinguishable ophthalmologically from that observed after injection of saline solution. This is in contrast to previous studies reporting considerable inflammatory reaction in animal eyes following injection of HA prepared from bovine vitreous, human umbilical cord, or rooster comb. 3 - 5 •8 •9 Quantitation of the inflammatory changes can be achieved by cell counts and chemical analysis of exudative proteins, provided that the specimen can be withdrawn without undue
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Vitreous
------------------·-
Injected Material Concentration of HA
--------
Saline HA I (BV) 2 mg/ml HA II (HUC) 10 mg/ml HA II (HUC) 18 mg/ml HA Ill (RC) 10 mg/ml HA IV (RC) 10 mg/ml HA V (RC) 10 mg/ml
N
Mean Protein Concentration mg/ml
4
5.7 (± 5.7)
7 (± 4)
4
0.25 (± 0.16)
13 (± 23)
2
2
4.4(±1.1)
29 (± 13)
2
0.29 (± 0.09)
1 (± 2)
2
6
7.3 (± 5.6)
5 (± 5)
4
0.26 (± 0.03)
1 (± 2)
2
2
8.6 (± 4.6)
8 (± 7)
2
0.31 (± 0.05)
0
2
4
6.3 (± 4.8)
40 (± 28)
4
1.06 (± 0.27)
42 (± 32)
2
4
2.0 (± 0.9)
5 (± 7)
4
0.58 (± 0.12)
6 (± 10)
2
4
34.8 (± 11.9)
1360 (± 1290)
4
4.6(±1.1)
Mean No. of Cells/mm 3
N
Mean Protein Concentration mg/ml
Mean No. of Cells/mm 3
----
1824 (± 976)
Clinical Grade -------
5-6
709
Table 3. Chemical Properties of Tested HA Preparations Preparation (Tissue Origin) 48-Hr Inflammatory Grade Concentration Intrinsic Viscosity HUG +2 10 mg/ml 2400 ml!g RC* +2 10 mg/ml 2200 ml/g RC +2 11 mg/ml 4400 ml/g RCt +5-6 10 mg/ml 2400 ml!g
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Amino Acid Composition ~-tg/mg HA Ala Gly Glu Ser
lieu
Leu
Asp
Thr
0.64
0.11
0.07
0.08
0.16
0.06
0.07
0.02
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0.03
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0.04
0.02
0.02
0.05
0.04
0.03
0.03
0.04
0.29
0.07
0.02
0.03
0.07
0.04
0.02
0.02
0.02
92% of injected HA was retained in vitreous at 48 hr. of injected HA was retained in vitreous at 48 hr.
t 94%
mechanical trauma. The owl monkey vitreous provides a very sensitive and reproducible biological test system because more than 90% of its vitreous volume is a collagen-freeH1 viscous liquid and not a rigid gel like that of other primates (except the bush baby) or the rabbit. Thus, the vitreous can be withdrawn and replaced with little or no mechanical damage, and the variation of the response to the same injected material is kept at a minimum. Clinical evaluation in combination with vitre- · ous and aqueous humor cell counts and Lowry protein determinations is necessary because of the high standard deviation of the protein exudation into the vitreous (average standard deviation ±22%) and especially the aqueous humor (average standard deviation ±52%). The combination of clinical and laboratory findings,
however, allows a reliable evaluation of the inflammatory potential of the particular HA batch on a grade + 1 to +6 scale provided that the preparation is· tested by injection into the vitreous. The HA preparations resulting in greater than a grade +2 reaction (corresponding to more than 50 cells/mm3 of vitreous and aqueous and an average of0.5 mg Lowry protein/ml vitreous and 5.7 mg Lowry protein/ml aqueous) should preferably not be used in human vitreous surgery. It must be emphasized, however, that even the severe inflammatory changes due to highly inflammatory (grade +5-6) HA injection are transient and do not cause late or lasting pathologic changes in the normal owl monkey eye up to two years after primary injection. The slight inflammation following injection of grade
Table 4. Aqueous Inflammatory Response to Primary Anterior Chamber Injection of Saline and HA
Treatment
N
Replaced Volume of Aqueous ml
Paracentesis Saline HA-(HUC) HA-(RC)
16 6 6 4
0.3 0.3 0.3 0.3
Hours after Injection 0 48 48* 48t
N = number of eyes. * 5% of injected HA was retained in aqueous humor at 24 hr. t 7% of injected HA was retained in aqueous humor at 24 hr. :j: 48-hr inflammatory reaction after intravitreous injection.
710
Mean Protein Concentration mg/ml 0.28 3.5 5.2 5.2
(± (± (± (±
0.21) 4.3) 1.9) 6.1)
Mean No. of Cells/mm 3
0
5 (± 5) 26 (± 14) 59 (± 36)
Clinical Grade in Anterior Segment 2 (2:j:) 2 (2:j:) 2 (5-6:j:)
+2 inflammatory HA, on the other hand, is acceptable for ocular surgery because it is comparable to the ocular response to saline. Injection of HA into the anterior chamber cannot serve as a test system for differentiation between grade + 2 and highly inflammatory HA because our data show that both preparations are almost equally well tolerated in the anterior segment. Therefore, this more convenient approach cannot substitute for intravitreous testing. The consequence, however, is that grade +5-6 inflammatory HA may be as safely used as grade + 2 inflammatory HA in surgery on the anterior segment (eg, implantation of intraocular lenses). Furthermore, HA injected into the anterior chamber does not diffuse into the vitreous, provided that the anterior hyaloid membrane is intact (Hultsch, unpublished data). The cause of the severe inflammation following intravitreous HA injection is not known and deserves further study; it is not caused by contamination with endotoxins. It appears that five minutes of autoclaving at l20°C reduces the inflammatory potential from a grade +5-6 to a grade +3 reaction. Unfortunately, this approach is not practical because it is accompanied by a significant depolymerization of HA (eg, from an intrinsic viscosity value of 2000 mVg to a value of 300 mVg). Exposure of the posterior segment (ciliary body?) is apparently a prerequisite for the development of inflammatory sequelae. As the present communication demonstrates, tissue origin, protein content, and amino acid composition are not correlated to the inflammatory potential. Thus, HA extracted from bovine vitreous, the rooster comb, and human umbilical cord may be equally well tolerated. Similarly, the inflammatory response to HA appears not to be correlated to the concentration used, provided that the preparation is of a low reactive grade. Thus, injection of an 18 rng/ml solution resulted in an inflammatory response comparable to that observed after injection of the lower concentrated (10 mg HA/ml) solution of the same umbilical cord batch. Regarding the inflammatory potential of different HA batches, preparations obtained from Med.-Chem. Products exhibited the highest variability, whereas those obtained from Pharmacia showed the lowest variability in inflammatory response. The chemical and inflammatory properties of HA preparations, packaged in glass syringes sealed in air-tight plastic bags, were unaltered over a period of five years. The higher molecular weight HA obtained from umbilical cord and rooster comb is more likely to be retained for longer periods of time in the human eye than the lower molecular weight bovine vitreous HA because the outflow of HA
from the vitreous depends on the molecular weight of the injected biopolymer. 11 Preliminary data obtained from post-open sky vitrectomy specimen (Hultsch and Schepens, unpublished data) indicate that in the human eye, injected HA is retained for significantly longer periods of time (up to 20 days) than in the owl monkey eye, which may explain the beneficial effect observed after the clinical use of low molecular weight HA (Etamucine)P A potential danger might be the development of glaucoma in predisposed human eyes; close monitoring of the intraocular pressure is therefore advisable. It is important to note that intraocular inflammation neither degrades HA nor results in an accelerated outflow from vitreous and aqueous. Similarly, experimental argon and krypton laser irradiation does not alter the structure of vitreous HA . 13 That is, laser coagulation therapy following intraocular HA injection will not diminish the tamponading effect of HA. The following characteristics of HA are of special importance for its use as intraocular implants and should be known to the ocular surgeon: (1) Concentration and molecular weight, both of which determine the rate of outflow from the eye via the anterior hyaloid membrane and the Schlemm's canal. Hyaluronic acid concentrations of 10-20 mg HA/ml solvent and intrinsic viscosity values of 2000-4000 mVg (preferably more than 3000 ml/g) will result in a long-lasting tamponading effect. (2) The inflammatory potential, which varies from HA batch to HA batch; it can be determined and quantitated by intravitreous testing only. The 48-hour inflammatory response should be tested in four eyes (preferably owl monkey eyes) and should not exceed the response to control injection of salinecorresponding to a grade + 1 - + 2inflammatory reaction on a grade + 1- +6 scale with less than 50 inflammatory cells/ml vitreous and aqueous, less than 0.5 mg Lowry protein/ml vitreous, and less than 6 mg Lowry protein/ml aqueous humor. Hyaluronic acid forms a gel only at a low pH (pH 2.5); the gel is therefore not usable for intraocular surgery. The viscoelastic fluidity of Na-hyaluronate at physiologic pH, on the other hand, makes the biopolymer easily injectable and provides a nonpermanent implant material suitable for repeated use. Our data on mechanisms controlling the macromolecular composition and regeneration of the vitreous in animal models14•15 provide a basis for the analysis of human material. Our experimental finding that HA reduces the influx of inflammatory proteins into the vitreous 11 indicates a regulatory function of HA; this may
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prove to be of additional importance for the use of HA in intraocular surgery. Of further importance for the retention of HA in the intraocular fluids is the presence of a novel factor, HADPF, in vitreous and aqueous humor. 16 HADPF (Hyaluronic Acid-Depolymerization Prevention Factor) inhibits the ascorbic acid-induced degradation of HA, thus reducing the outflow of injected HA.
ACKNOWLEDGMENT
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The assistance of J. W. Appleby and B. Pietrasz and the critical review of the manuscript by Dr. Alice Adler are gratefully acknowledged.
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REFERENCES 1. Deutschmann R. Ueber ein neues Heilverfahren bei Netzhautabl~sung. Beitr Augenheilkd 1895; 20:1-80. 2. Peyman GA, Ericson ES, May DR. A review of substances and techniques of vitreous replacement. Surv Ophthalmol 1972; 17:41-51 . 3. Constable IJ, Swann DS. Biological vitreous substitutes. Inflammatory response in normal and altered animal eyes. Arch Ophthalmol 1972; 88:544-8. 4. Widder W. Hyalurons aure als GlaskDrperimplantat bei Netzhautablosung. Albrecht von Graefes Arch Klin Exp Ophthalmol 1960; 162:416-29. 5. Kl~ti R. Die heutigen Probleme in der Amotio-chirurgie. Ophthalmologica 1968; 156:415-24.
6. Bitter T, Muir HM. A modified uronic acid carbazole reaction. Anal Biochem 1962; 4:330-4. 7. Peczon BD, Antreassian R, Bucay P. Chromatographic analysis of hydroxylysine glycosides and acid hydrolysates. J Chromatogr 1979; 169:351 -6. 8. Balazs EA, Sweeney DB. The injection of hyaluronic acid and reconstituted vitreous into the vitreous cavity. In: McPherson A (ed.). New and Controversial Aspects of Retinal Detachment. New York: Hoeber Medical Division, Harper & Row, 1968; 371-6. 9. KIDti R. Hyaluronsaure als GlaskDrpersubstituent. Ophthalmologica 1972; 165:351-9. 10. Chakrabarti B, Hu ltsch E. Owl monkey vitreous: a novel model for hyaluronic acid structural studies Biochem Biophys Res Commun 1976; 71:1189-93. 11. Hultsch E. Vitreous structure and ocular inflammation. In Silverstein AM, O'Connor GR (eds.). Immunology and Immunopathology of the Eye. New York: Masson, 1979; 97-103. 12. Edmund J. Vitreous substitute in the treatment of retinal detachment. Mod Probl Ophthalmol 1974; 12:370-7. 13. Hultsch E, Ducrey N. Effect of argon and krypton laser irradiation on the normal owl monkey vitreous. Mod Probl Ophthalmol1979; 20:184-7. 14. Hultsch E. Regeneration of hyaluronic acid in the owl monkey vitreous: evidence for control mechanisms. ARVO 1978. Invest Ophthalmol Vis Sci April, 1978; Suppl: 209. 15. Pietrasz B, Hultsch E. Pathophysiology of vitreous hyaluronic Acid. ARVO 1980. Invest Ophthalmol Vis Sci April, 1980; Suppl: 232. 16. Hultsch E, Mukherjee DC. Studies on the comparative deopylmerization behavior of native and purified hyaluronic acid by ascorbic acid oxidation. Fed Proc 1980; 39:1637.