Keywords
chondral lesion - collagen - knee - rats - treatment
Introduction
The treatment of chondral lesions remains a challenge for orthopedists because of
the low regenerative ability of the cartilaginous tissue.[1]
[2]
These injuries result from several metabolic, genetic, vascular, and/or traumatic
factors. Their classification considers the size and thickness of the affected cartilage.[3]
[4] Its actual incidence remains unknown, since most of these injuries are asymptomatic.
Therefore, many patients do not seek medical help, even when the disease is in its
more advanced stages. Chondral lesions are common joint conditions, especially in
elderly and obese subjects, causing pain, stiffness, and functional limitation. Joints
under mechanical overload are the most compromised, particularly the knee.[3]
It is estimated that more than 10% of the population over 60-years-old have severe
joint issues due to some degree of articular impairment.[4] Senna et al.[5] and Henrotin et al.[6] reported a prevalence of 4.14% in the Brazilian population. Moreover, radiological
studies show changes in 30% of men and women over 65-years-old; among them, one-third
is symptomatic.
Despite the role of mechanical injuries in articular cartilage degradation, the inflammatory
process plays a critical role in the pathophysiology of the disease, triggering it
or accelerating its evolution.[4] The exact sequence of pathological events remains poorly understood, making consensus
on the best therapeutic approach difficult. There is still no cure, and treatments
only alleviate symptoms.[7]
Recent oral treatments with nutraceutical agents have a weak scientific recommendation.
This supplementary treatment consists of using laboratory-produced nutrients to strengthen
the articular cartilage and delay or prevent the evolution of the disease.[6]
In this context, the hydrolyzed collagen resulting from the hydrolysis of crude collagen
fibers or powder stands out. It has a proven role in maintaining and reconstituting
the skin, bones, cartilage, and extracellular matrix. Hydrolyzed collagen is safe
and has minimal adverse effects. Its amino acid composition is rich in proline, which
preferentially accumulates in cartilage and presents a chondroprotective effect.[6] Factors such as aging and a poor diet can affect the demand for collagen in the
body, contributing to the risk of bone and joint dysfunctions, and the eventual need
for dietary supplementation.[5]
[7]
[8]
Additionally, it is possible to isolate collagen peptide fragments through enzymatic
hydrolysis.[6] Collagen peptides have bioactive properties and may direct the production of specific
proteins, scavenge free radicals, prevent lipid oxidation, and act as chelators for
transition metals. As food supplements, they have a role in skin aging and osteoporosis
prevention.[9]
Some studies demonstrated the role of nutraceuticals by comparing the time of chondral
injury evolution, prevention, or both, between two groups with knee chondral injury,
the first one being treated with oral hyaluronic acid and an exercise program, and
the second one submitted only to physical exercises. At a 1-year follow-up, the first
group showed a different outcome, with fewer symptoms and lower use of analgesics.[10]
[11]
[12]
Thus, this study evaluates the effects of hydrolyzed collagen and collagen peptide
in treating chondral lesions 30 days after their induction in the knee articular cartilage
of rats.
Method
Ethical Considerations
This research followed the precepts of the Brazilian legislation regarding animal
husbandry and use (Federal Law number 11.794, of 2008) and the standards from the
Brazilian College of Animal Experimentation. The Ethics Committee on the Use of Animals
of the Center for Biological and Health Sciences from Universidade do Estado do Pará
(UEPA) approved this research project under protocol number 09/2019.
Study Type
This experimental study used 18 male, 120-day-old Rattus norvegicus from the Wistar strain, weighing approximately 240 g. The rats came from the animal
facility of Instituto Evandro Chagas. They were placed in plastic cages and kept in
a controlled environment for temperature, humidity, light, and noise.
We divided the animals into three groups, each with 6 subjects: the control group
(CG), collagen peptide group (CPG), and hydrolyzed collagen group (HCG). Drug doses
for each group were based on previous studies published in the literature.[10]
[11]
[12]
[13]
Surgical Procedures
The articular damage was induced across the patellar ligament through a single intraarticular
infiltration of a solution containing 2 mg of sodium iodoacetate, in a total volume
of 25 μl in the right knee joint of previously anesthetized rats, with a flexed at
a 90-degree angle, using a 26G X 3/8-gauge needle.
Treatment
Anesthesia for subsequent knee infiltration consisted of intraperitoneal injection
of ketamine and xylazine at doses of 70 mg/kg and 7 mg/kg, respectively.[14] A booster, if required, consisted of an intramuscular application of ¼ of the initial
dose.
After joint injury induction, all animals received analgesia with intraperitoneal
tramadol at a 2 mg/kg dose. They were kept in cages identified by groups, with food
and water ad libitum, for 30 days.
Animals from the CG (n = 6) received no treatment after the chondral lesion.
Those from the CPG (n = 6) received collagen peptide (0.16 g/kg/day) by gavage after
chondral lesion induction.
Animals from the HCG (n = 6) received hydrolyzed collagen (0.14 g/kg/day) by gavage
after chondral lesion induction.
The vehicle for component dilution was purified water. The rats were weighed daily
for potential dose adjustments.
Histological Processing
The animals were euthanized by anesthetic overdose. The right knee was dissected from
the hip to the ankle region, leaving the joint capsule intact. The samples were fixed
in 10% formalin and submitted to histopathological analysis. The knees were kept in
this solution for 1 day and demineralized in 5% nitric acid for 2 to 3 days. Tissues
were embedded into paraffin blocks to prepare tibial sections for staining with hematoxylin-eosin
and the Masson trichrome stain.
The histopathological analysis consisted of chondrocyte clusters counts in the superficial
and intermediate layers of the cartilage. The Osteoarthritis Cartilage Histopathology
Assessment System (OARSI) classified the depth of the lesion in six degrees, and the
extension of the joint surface lesion in four stages ([Fig. 1]).
Fig. 1 Histopathological analysis. Group 1: normal articular cartilage. Group 2: joint surface
discontinuity. Group 3: formation of fissures or gaps on the joint surface. Group
4: erosion of the articular cartilage surface. Group 5: denudation of articular cartilage.
Group 6: deformation of the subchondral bone (reproduced from Pritzker et al., 2006).
The extent of the articular cartilage lesion determined the stage of chondral injury
([Table 1]).
Table 1
|
Stage
|
% of involvement (surface, area, volume)
|
|
Stage 0
|
No osteoarticular activity
|
|
Stage 1
|
< 10%
|
|
Stage 2
|
10–25%
|
|
Stage 3
|
25–50%
|
|
Stage 4
|
> 50%
|
Next, we crossed the data on groups and injury stages in a table to determine the
grade of a standardized score ([Table 2]).
Table 2
|
Grades
|
Stages
|
|
E1
|
E2
|
E3
|
E4
|
|
G1
|
1
|
2
|
3
|
4
|
|
G2
|
2
|
4
|
6
|
8
|
|
G3
|
3
|
6
|
9
|
12
|
|
G4
|
4
|
8
|
12
|
16
|
|
G5
|
5
|
10
|
15
|
20
|
|
G6
|
6
|
12
|
18
|
24
|
The Kolmogorov-Smirnov test assessed normality. Statistical analysis of the study
data used the Kruskal-Wallis test. If there was a statistically significant difference,
the Student-Newman-Keuls test was used, adopting a significance level of α = 0.05.
Analysis was performed with the BioEstat 5.4 software.
We used Word 2016 (Microsoft Corp., Redmond, WA, USA) to prepare this manuscript,
Excel 2016 (Microsoft Corp., Redmond, WA, USA) to organize and construct tables and
graphs, and PowerPoint 2016 (Microsoft Corp., Redmond, WA, USA) to create the slide
show.
Results
[Table 3] shows the OARSI classification. Initially, the Lilliefors test verified data normality
and revealed a nonparametric distribution. Then, we applied a Kruskal-Wallis test
that detected a difference among groups (p = 0.0008). The Student-Newman-Keuls test confirmed this difference.
Table 3
|
Rats
|
Control Group
|
Hydrolyzed Collagen Group
|
Collagen Peptide Group
|
|
Group
|
Stage
|
Score
|
Group
|
Stage
|
Score
|
Group
|
Stage
|
Score
|
|
R1
|
2
|
1
|
2
|
2
|
1
|
2
|
2
|
1
|
2
|
|
R2
|
4
|
1
|
4
|
1
|
1
|
1
|
1
|
1
|
1
|
|
R3
|
3
|
1
|
3
|
2
|
1
|
2
|
2
|
1
|
2
|
|
R4
|
2
|
2
|
4
|
1
|
1
|
1
|
2
|
1
|
2
|
|
R5
|
4
|
1
|
4
|
2
|
1
|
2
|
2
|
1
|
2
|
|
R6
|
2
|
2
|
4
|
2
|
1
|
2
|
2
|
1
|
2
|
|
Mean
|
2.83
|
1.33
|
3.5
|
1.66
|
1
|
1.66
|
1.83
|
1
|
1.83
|
The OARSI classification proposed by Pritzker et al.[15] revealed the correspondence among groups (CG, HCG, and CPG; p < 0.05) and the statistical difference between HCG and CPG (p = 0.3632) ([Fig. 2]).
Fig. 2 Graph of the correspondence of the joint injury groups and each rat from the experimental
groups (p < 0.05 for all groups; p = 0.3632 when comparing HCG and CPG).
There was no statistically significant difference between the groups regarding stages
of injury (p = 0.11) ([Fig. 3]).
Fig. 3 Graph of the correspondence of joint injury stages and each rat from the experimental
groups (CG, HCG, and CPG) (p = 0.11).
Regarding the scores, there was a statistical difference when comparing HCG and CPG
to CG (p < 0.05) ([Fig. 4]). The scores were 1.66, 1.83, and 3.5 for HCG, CPG, and CG, respectively.
Fig. 4 Graph of the correspondence of joint injury scores and each rat from the experimental
groups (CG], HCG, and CPG) (p < 0.05).
[Table 4] shows the number of chondrocyte clusters in the superficial and intermediary layers
of the cartilage.
Table 4
|
Cartilage layer
|
Control group
|
HCG
|
CPG
|
|
Superficial 12h
|
2 to 3
|
3 to 4
|
3 to 4
|
|
Intermediary 12h
|
4 to 5
|
4 to 5
|
4 to 5
|
Discussion
The literature supports the beneficial effect of the oral administration of hydrolyzed
collagen in joint diseases. A double-blinded, randomized, clinical study demonstrated
that treatment with 5 g of collagen peptides twice a day in patients with chondral
injury of the knee promoted a statistically significant reduction in Western Ontario
McMaster Universities Osteoarthritis Index (WOMAC) scores, a quality-of-life questionnaire
specific to this population.[15]
Our study revealed an improvement in the induced knee injury, indicating that the
compounds reached the chondrocytes and with a proper recognition by their surface
proteins. The literature reports the accumulation of hydrolyzed collagen in murine
joints. The beneficial action of hydrolyzed collagen may be associated with the stimulation
of chondrocyte metabolism, activating collagen biosynthesis.[7]
There was no difference among groups regarding the number of chondrocyte clusters
in the superficial and intermediary layers of the articular cartilage, except for
CG, in which the superficial layer presented fewer cells. This finding contradicts
the literature, which describes the classic histological pattern of cartilage affected
by joint injury with an increase of cell clusters in both size and number.[7]
[16]
[17] The experimental model of injury induction may justify this finding. The fact that
joint damage results from an external agent, instead of an intrinsic issue with the
balance between anabolism and cartilage catabolism, may account for this discrepancy.
Furthermore, although apoptosis is a component of the local response to injury, the
decrease in cluster count may be related to it, either as an inducing factor or a
product of osteoarticular degeneration.
There was a statistically significant difference in the classification of histological
groups and lesion scores (p < 0.05). This finding suggests a delay in the inherent inflammatory process and the
stabilization of the extracellular matrix degradation due to an increased synthesis
of its components by chondrocytes. An experimental study in bovine and human chondrocyte
models reports that treatment with hydrolyzed collagen inhibited the production of
inflammatory mediators (e.g., nitric oxide and prostaglandin E-2). This inhibition
occurred even in the presence of proinflammatory cytokines (interleukin β-2). Additionally,
the treatment decreased the production of metalloproteinases (which degrade the extracellular
matrix) and the expression of the enzyme cyclooxygenase 2. All these findings were
more prominent in human chondrocytes.[18]
[19]
On the other hand, a metanalysis by Bakilan et al.[20] concluded that there was no evidence the oral administration of hydrolyzed collagen
reduced cartilage destruction. It is noteworthy, however, that this study considered
clinical trials alone. The authors stated that the oral administration of type II
collagen is a new treatment with the potential to prevent joint destruction, pain,
and loss of function, warranting further studies.[18]
[21]
[22]
There was no difference between the results from HCG and CPG, which is consistent
with the results from a randomized clinical trial by Kumar et al.[23] Therefore, despite the origin of collagen peptides, their efficacy remains the same
in our study.
For humans, pain reduction indirectly indicates an improvement in the joint conditions
of patients with knee chondral injury. Moreover, it may be associated with the initiation
of the repair process by collagen peptide accumulation in the cartilaginous tissue.
Accumulated collagen helps maintain cartilage structure and function.[24]
Regarding lesion stage (extension), there was no statistically significant difference
among groups. In fact, stage 1 injury prevailed even among CG specimens. We can interpret
this finding according to a study analyzing chondral lesion defects in human knees
with spectroscopy and histopathology. In this report, Spahn et al.[24] showed that the cartilage surrounding the defect and the remaining joint presented
changes even if its appearance was intact. Thus, the entire joint is compromised by
degeneration even though it remains histologically unchanged to a considerable extent.[20]
In preclinical studies, 24% of patients with joint damage who received 5 to 7 g of
hydrolyzed collagen orally experienced substantial improvement with complete absence
of pain, and 44% reported remarkable improvement.[11] Despite the noteworthy findings and the lack of side effects, this study had no
statistical analysis.[11] There were few side effects descriptions in humans taking more than 10 g/day. The
most common side effects included headaches and mild gastrointestinal disorders, and
their presence did not contraindicate the treatment.
We cannot conclude whether oral collagen administration is effective in cases with
higher joint surface wear. However, preclinical research indicates that the effects
of collagen supplementation in improving joint damage are dose- and time-dependent.
As such, future investigations may contribute to elucidating these data, with longer
treatment times for the objective evaluation of the long-term evolution of the disease
with the use of the studied therapeutic interventions.
Conclusion
The proposed treatment of the induced chondral lesion with the oral administration
of hydrolyzed collagen and collagen peptides was effective. It resulted in stabilization
or regression of the lesion, deserving further experimental studies to understand
and improve the primary outcome of our study.
