Establishment of Vasculature in Hyper-Crosslinked Carbohydrate Polymer as Scaffolding for Tissue Engineering and Regeneration

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Effective tissue repair and regeneration hinge on the establishment of a functional vascular network capable of delivering oxygen, nutrients, immune cells, and signaling molecules essential for intrinsic healing processes. In the context of bone grafts, substitutes, and engineered constructs, vascularization is particularly critical to prevent hypoxia-induced cell death and graft failure, especially in large or critical-sized defects. This study evaluated the vascularization potential of a novel hyper-crosslinked carbohydrate polymer (HCCP) bone graft substitute relative to hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP), a widely used clinical comparator. Results demonstrated that HCCP significantly enhanced neovascular infiltration and reduced immunogenic response when compared with HA/β-TCP. These findings support HCCP’s potential as a next-generation bone graft material, particularly for applications requiring rapid vascular integration and low inflammatory burden.

Abstract

Vascularization is one of the key components of tissue engineering and must accompany the ingrowth of new tissues to establish an environment conducive to repair and regeneration of damaged tissue. The overarching objective of this study was to investigate whether the hyper-crosslinked carbohydrate polymer (HCCP) could promote the establishment of new vasculature compared to hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP), which is widely used in orthopedic procedures. Sprague Dawley rats (n = 12) were implanted subcutaneously with HCCP or HA/β-TCP and evaluated histologically for the ingrowth of new vasculature at 3, 14, and 28 days post-implantation. HCCP showed significantly greater levels of vascularization when compared to HA/β-TCP at all time points evaluated (p < 0.05). HA/β-TCP showed transient inflammation at 14 days post-implantation, whereas minimal immune activities were noted in HCCP. These findings suggest that HCCP promotes the establishment of new vasculature without a significant immune response.

1. Introduction

Bone infections, tumors, and fractures caused by trauma or osteoporosis are the main causes of bone defects requiring surgical repair [1]. Typically, allografts, autografts, or bone graft substitutes (BGS) are used in reconstructive orthopedic surgeries to bridge skeletal gaps and facilitate bone regeneration in defects with impaired healing capacity, such as in the case of nonunion, malunion, and avascular necrosis [2]. Autografts are currently the gold standard due to their osteogenicity, intrinsic histocompatibility, and the absence of disease transmission risk [3]; however, their use is compromised by donor site morbidity, limited availability, and increased operation complexity [4,5]. Allografts pose additional risks of disease transmission, infection, immunogenicity, and immune rejection [6]. To avoid these complications, BGS composed of materials such as bioactive glasses, hydroxyapatite (HA), beta-tricalcium phosphates (β-TCP), calcium sulfates, and polymer-based scaffolds [7] have emerged as alternatives for bone repair. These osteoconductive materials serve as scaffolding for new bone growth that is perpetuated by the native bone adjacent to a skeletal defect site. Scaffolding enables osteoblasts and bone progenitor cells from the bone defect margin to infiltrate the graft framework, generating new bone that bridges the skeletal gap [8]. Osteoprogenitor cell infiltration into the scaffold is mediated by the vascular endothelium [9,10,11], and osteogenic repair is dependent on angiogenesis at the defect site to deliver cytokines, hormones, growth factors, and osteoblasts that are necessary for bone formation [12,13].

Poor vascularization of the graft framework leads to atrophic non-union, excessive fibrotic scar tissue formation, and impairs osteogenesis [14,15,16]. Of the three types of bone non-union (atrophic, hypertrophic, and oligotrophic), atrophic non-union, owing to deficient angiogenesis at the bone defect site, is the primary cause of poor fusion outcomes [17,18]. Atrophic non-union results from insufficient blood supply [19] and is the most recalcitrant to treatment, often requiring multiple surgeries over many years [20]. To address these issues, porous polymer-based materials and hydrogels have been considered due to high biodegradability, antifibrotic [21] and pro-angiogenic properties [22,23], yet their ability to promote angiogenesis in the repair of critical-sized bone defects compared to crystalline BGS, such as calcium sulfates, β-TCP, and HA, has not been well-studied.

This study was conducted to compare the angiogenic properties of Hyper-Crosslinked Carbohydrate Polymer (HCCP), an emerging carbohydrate polymer-based, non-mineralized, synthetic BGS [24], to a widely used BGS comprising bioglass plus 60% HA/40% β-TCP (HA/β-TCP). HCCP has been shown to support bone regeneration and repair with exceptional porosity, degradation rate, biocompatibility, and is an FDA-cleared BGS in non-weight bearing applications. In addition to angiogenesis, this study compared the local tissue response, cell migration and adhesion, and early stages of degradation of HCCP to HA/β-TCP after dorsal subcutaneous implantation in rats.

2. Materials and Methods

2.1. Animals

The Sprague Dawley rat model for ectopic bone formation via subcutaneous BGS implantation has been previously established [25]. Healthy male Sprague Dawley rats (n = 12; 350–400 g; 10–12 wks. old) sourced from Charles River Laboratories (Wilmington, MA, USA) were housed individually and provided with purified tap water and laboratory-certified, pelleted rodent feed (LabDiet #5002) ad libitum. Primary animal housing was maintained at 22 ± 4 °C. All animal procedures were performed in accordance with Good Laboratory Practice (GLP), the Animal Welfare Act, and were approved by the Institutional Animal Care and Use Committee (IACUC).

2.2. Surgical Procedures

Following an acclimation period of at least 7 days, rats were randomized using a Microsoft Excel–generated randomization scheme based on the RAND() function. Unique animal IDs were entered into Excel, a RAND() value was generated for each animal ID, and animals were assigned to study timepoint groups (3-day, 14-day, and 28-day; four animals per group) according to the resulting randomized order. Animals were sedated with a mixture of ketamine hydrochloride (75 mg/kg) and midazolam (4 mg/kg), in addition to a pre-operative subcutaneous dose of extended-release buprenorphine XR (0.65 mg/kg), for analgesia. The dorsum of each animal was shaved, and surgical plane anesthesia was induced and maintained using isoflurane (0.25–4%) volatilized with pure oxygen (2 L/min) delivered through a precision vaporizer. Surgical sites were thoroughly prepared with a 2% chlorhexidine gluconate scrub and sterile draped with the animal in the prone position. Topical ophthalmic ointment was also applied to conjunctiva of each eye. Core body temperature was continuously monitored and maintained intraoperatively using a PhysioSuite Small Animal Physiological Monitoring System (Kent Scientific, Torrington, CT, USA) with integrated Temperature Monitor and Homeothermic Control Module. The PhysioSuite’s temperature sensor rectal probe was used to provide real-time body temperature readouts. Normothermia was maintained using the system’s integrated heating pad positioned under the animal during surgery, with heat output adjusted as needed to keep body temperature within physiologic limits throughout the procedure.

Each animal received six subcutaneous implants placed alongside the dorsal midline, with each animal randomly receiving three test implants (HCCP) on one side and three predicate implants (HA/β-TCP) on the contralateral side. Skin incisions measuring ~1.5 cm were created at each of the six implant sites, ~1 cm lateral to the dorsal midline and ~2 cm distal to other implant sites. Subcutaneous pockets (>10 mm from incision line) were created by blunt dissection, with one implant placed per pocket. HCCP or HA/β-TCP was hydrated with 0.1–1 mL physiologic saline and implanted in disc form (10 mm diameter, 2–3 mm thickness). Each incision was sutured with 4-0 PDS-II absorbable suture (Ethicon, Cincinnati, OH, USA) and reinforced with surgical tissue adhesive.

Routine clinical observations were conducted daily postoperatively. Enrofloxacin (5 mg/kg, per os) was provided 7 days postoperatively for antibiotic prophylaxis for infection control. Carprofen (5 mg/kg, subcutaneous) and buprenorphine XR (0.65 mg/kg, subcutaneous, single dose) were administered for inflammation and pain management, respectively. Post-recovery, animals were monitored daily for incision healing, skin condition at the treatment sites, mobility, and activity level. Animals were euthanized in a CO₂ chamber in accordance with the AVMA Guidelines on Euthanasia, at 3-day, 14-day, and 28-day endpoints.

2.3. Histology

Excised implants were measured and photographed, then fixed in 10% neutral buffered formalin for 24 h before transfer to 70% ethanol. Fixed specimens were submitted to VDx Veterinary Diagnostics (Davis, CA, USA) for histological processing. The explants were embedded in paraffin, thin sectioned (4–7 μm thickness) at two different levels within the block and stained with hematoxylin and eosin (H&E). Sections of each implant were examined microscopically (Olympus BX51 Fluorescence Microscope, Olympus Corporation, Tokyo, Japan) for angiogenesis, inflammatory response, and fibrosis. Vascular assessment was based on the identification and quantification of lumenized blood vessels, reflecting established neovascular infiltration rather than early, non-lumenized stages of angiogenesis. Draining local lymph nodes (axillary and inguinal) were collected for paraffin embedding and staining with H&E, then evaluated microscopically for atypical immune response.

2.4. Color Image Processing and Channel Analysis

Gross images of subcutaneous implants were taken prior to excision and analyzed with ImageJ’s (version 1.54g) color deconvolution processing software. RGB (Red, Green, Blue) color channels were split, and red channel intensity color codes for the region of interest (circular region 5 mm in diameter manually selected for each implant) were extracted. Color codes were tabulated for analysis and controlled using adjacent subcutaneous layer red channel intensity. The red channel was used as a surrogate for blood content based on the optical properties of hemoglobin. Regions of interest (ROIs) were defined for the implant and for adjacent subcutaneous tissue (background control). For each ROI, mean red-channel intensity (8-bit; 0–255) was measured. Implant red-channel intensity was normalized to adjacent tissue by calculating the difference between implant and background mean intensity values (implant − background), yielding relative intensity values for comparison across samples. Negative values indicate that the implant region was lighter (lower red-channel intensity) than surrounding tissue.

2.5. Vascular Density Analysis

Quantification of vascularization within the implant sections was performed on H&E-stained slides obtained at 3-, 14-, and 28- days post-implantation by two independent evaluators blinded to treatment group. For each implant, two representative sections were analyzed at different levels. Using ImageJ, individual blood vessels were segmented manually based on lumen structure and cellular morphology, and total vascular area was measured for each section (lumens smaller than 10 μm in diameter were excluded from analysis). The total tissue area for each implant section was also recorded. Fractional vascular area was defined as the ratio of cumulative vascular area to total implant area. Data from replicate implants were first averaged at the individual animal level. For each animal, measurements from the three HCCP implants and the three HA/β-TCP implants were averaged separately to generate one material-specific value per animal at each time point. These animal-level means (n = 4 animals per time point) were then used for all group-level statistical analyses, and statistical significance was determined using two-way ANOVA with Tukey’s Honestly Significant Difference (HSD) test (RStudio, version 2024.12.0+467). A p-value < 0.01 was considered highly significant.

2.6. Statistical Analysis

All results are reported as mean  ±  standard error of the mean (SEM) and calculated using Microsoft Excel software (Microsoft). Paired two-sided Student’s t-test were used for within-animal comparisons between HCCP and HA/β-TCP for each time point. Two-way analysis of variance (ANOVA) was used to assess the effects of implant type (HCCP versus HA/β-TCP) and time points. Tukey’s Honestly Significant Difference * HSD) post hoc test was utilized to control for multiple comparisons when significant defects were noted. Statistical significance (p ≤ α, α = 0.05) was determined by analysis of variance or two-sided Student’s t-test analysis. Two-way ANOVA with replication and Tukey’s HSD Test adjusted p-values were calculated using RStudio (Posit, PBC).

3. Results

3.1. Clinical Observations

All animals (n = 12) that received subcutaneous HA/β-TCP and HCCP implants underwent the surgical procedures without any adverse clinical episodes. Animals in both groups displayed normal appetite, movement, incision healing, skin condition, mobility, and activity level. Animals remained in good overall health throughout the study period.

3.2. Gross Morphology

HA/β-TCP (Figure 1A,C,E) implants exhibit minimal darkening at 2 (Figure 1C) and 4 weeks (Figure 1E) post-implantation, in addition to an inconsistent, speckled appearance at 4 weeks post-implantation. HCCP (Figure 1B,D,F) implants exhibit progressive darkening at 2 (Figure 1D) and 4 weeks (Figure 1F) post-implantation, with evidence of blood infiltration and vascularization within the implant. The degree of darkening observed at 2 and 4 weeks was similar to a vascularized tissue such as muscle.

3.3. Implant Volume and Vascularity

Changes in volume of the implants were quantified post-implantation at all time points (Figure 2). Implant volume was assessed by manually measuring implant diameter and thickness. Cylindrical approximation was then used to estimate volume. Both implant types showed a significant initial reduction in volume 3 days post-implantation when compared to pre-implantation; HCCP exhibited a 19.2 ± 3.0% reduction, and HA/β-TCP exhibited a 10.9 ± 1.8% reduction in implant volume. The volume of HA/β-TCP implants continued to decline over time, with a 17.6 ± 4.0% reduction in volume at 14 days and a 31.1 ± 3.6% reduction at 28 days (p_adj = 0.0057). In contrast, the volume of HCCP increased significantly by 10.1 ± 5.9% between 3 days and 28 days after implantation (p_adj = 0.0069). At 28 days, the HCCP implant was significantly larger than the HA/β-TCP implant by 22.0 ± 6.5% (p_adj = 1.5 × 10⁻¹²).

Quantitative assessment of blood perfusion into implant materials was performed using red channel intensity (IV) analysis, leveraging the light absorption and reflection properties of hemoglobin in the visible red spectrum as an indirect measure of vascularization. Gross changes in implant vascularity over time post-implantation were analyzed by comparing red color intensity values across groups (Figure 3). HCCP showed significantly greater red channel intensity, measured by 8-bit color depth, at all time points, indicating enhanced vascular infiltration. At 3 days post-implantation, HCCP showed a mean intensity of 67.5 ± 4.2 IV, markedly greater than HA/β-TCP (−8.3 ± 4.5 IV; p < 0.0001). This trend persisted through 28 days, with HCCP reaching 117.8 ± 4.3 IV at day 14 versus 22.2 ± 5.2 IV in HA/β-TCP (p < 0.001), and 94.2 ± 6.7 IV at day 28 compared to 18.6 ± 3.2 IV in HA/β-TCP (p < 0.001). Both materials demonstrated significant increases in red channel intensity from day 3 to days 14 and 28 (p < 0.001), though the magnitude of change was consistently greater in HCCP, supporting its superior vascularization profile.

Quantitative histological analysis showed no detectable vascularization in either group at 3 days post-implantation (Figure 4, fractional vascular area = 0.0000). At 14 days, HCCP exhibited significantly greater vascularization than HA/β-TCP (0.0777 ± 0.0067 vs. 0.0022 ± 0.0003; p < 0.001). At 28 days, HCCP again showed significantly higher vascular area (0.1262 ± 0.0141) compared to HA/β-TCP (0.0036 ± 0.0004; p < 0.001). Within-group comparisons showed significant increases in HCCP from day 3 to 14 (p < 0.001) and 14 to 28 (p < 0.001); HA/β-TCP also increased slightly over time (day 3 to 14: p < 0.01 day 14 to 28: p < 0.01).

3.4. Histological Evaluation of Implant Sites

Histological analysis revealed no evidence of cellular infiltration in either implant material at 3 days post-implantation (Figure 5A,B). By day 14, both HCCP (Figure 5C) and HA/β-TCP (Figure 5D) exhibited notable increases in cellularization, though the extent and quality of vascular development differed markedly. HCCP showed definitive blood vessels at both 14 and 28 days (Figure 5D,F), characterized by branching venules and clearly defined endothelial linings within the scaffold pores (Figure 6). In contrast, HA/β-TCP showed fewer blood vessels, often filled with red blood cells, at corresponding time points (Figure 5C,E). Notably, HA/β-TCP implants at day 14 displayed myofibrinous degenerative changes, moderate inflammation, and myofibrous cellular ingrowth, suggesting a less favorable remodeling profile. HCCP histology remained consistent between days 14 and 28 with minimal inflammatory response and the presence of multinucleated giant cells adjacent to the material (Figure 7). Both implant types exhibited minimal structural degradation throughout the study period (Figure 5, black arrows), indicating material stability in vivo.

4. Discussion

The comparative performance of HCCP and HA/β-TCP in promoting vascularization reveals key mechanistic differences rooted in scaffold architecture, immune modulation, and material resorption dynamics. These distinctions are critical for understanding the design parameters that underpin successful bone tissue engineering and regenerative scaffold integration.

4.1. Scaffold Architecture and Angiogenic Infiltration

The enhanced vascularization observed in HCCP implants can be attributed to its highly porous, interconnected microarchitecture, which optimizes endothelial migration, oxygen diffusion, and capillary formation. Pore size in the range of 200–400 μm is considered optimal for angiogenesis, as it allows for both cellular infiltration and the formation of patent, lumenized blood vessels [26,27]. HCCP falls within this geometrical regime, promoting robust vascular invasion. In contrast, HA/β-TCP, with its denser, crystalline structure and lower interconnectivity, creates a diffusion barrier and restricts deep tissue penetration, thereby delaying neovascularization [18,28]. This study focused on the quantification of lumenized blood vessels to compare how HCCP and HA/β-TCP support the establishment of mature vasculature within the implant environment. As such, the analyses were designed to capture established neovascular infiltration rather than the earliest, pre-lumenized stages of angiogenesis. While the differences between HCCP and HA/β-TCP in neovascularization were clearly demonstrated in this study, future investigations incorporating endothelial-specific markers (e.g., CD31 or von Willebrand factor) may provide additional insight into early angiogenic events and vessel maturation dynamics.

Moreover, interconnectivity, the degree to which pores are spatially connected, is a critical but often underappreciated factor in scaffold performance. High interconnectivity in HCCP facilitates the establishment of a continuous vascular network, which is vital for nutrient delivery, waste removal, and cellular migration [29,30]. These findings are consistent with previous studies that demonstrate enhanced endothelial tube formation and anastomosis in scaffolds with high porosity and pore interlinking [31].

The gross tissue observations and histological vascular area quantifications support this architectural influence (Figure 1 and Figure 4). HCCP implants showed substantial red color intensity (Figure 3) and significantly increased fractional vascular area as early as 14 days post-implantation, further accentuated by 28 days (Figure 4). These changes coincide temporally with neovascular expansion and confirm the role of scaffold porosity in supporting capillary infiltration, red blood cell perfusion, and subsequent tissue viability.

The HA/β-TCP implant material used in this study is a mineral-based graft composed of hydroxyapatite and β-tricalcium phosphate with a predominantly crystalline architecture designed to provide osteoconductive support [32]. Such materials typically exhibit relatively rigid pore structures with limited interconnectivity and rely primarily on surface-mediated protein adsorption to support tissue ingrowth. In contrast, HCCP is a synthetic carbohydrate-based scaffold modeled after the glycan-rich component of native proteoglycans, resulting in a highly porous, interconnected architecture and a hydrated polymer network [24]. This glycan-like chemistry may facilitate growth factor binding, retention, and presentation, in addition to supporting cellular infiltration and vascular ingrowth [33]. These compositional and structural differences provide a plausible mechanistic basis for the enhanced vascularization observed in HCCP compared with HA/β-TCP.

4.2. Degradation Profiles and Immune Modulation

Degradation rate and byproducts of biomaterials shape the local immune microenvironment, impacting both inflammation and regenerative progression. HA/β-TCP degrades asymmetrically: β-TCP dissolves rapidly via passive hydrolysis, releasing phosphate and calcium ions that can perturb the local ionic balance, acidify the microenvironment, and activate macrophages [34,35]. This can induce a pro-inflammatory M1 macrophage phenotype, characterized by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) release, which inhibits endothelial stability and vessel maturation [36]. The histological presence of widespread inflammatory infiltrates at HA/β-TCP sites by day 14, and persisting at day 28 (Figure 7A), supports this mechanism.

In contrast, HCCP exhibits slower, more uniform hydrolytic degradation that avoids local pH extremes and releases carbohydrate monomers that are less immunostimulatory [37]. Histological findings of only mild inflammation and the presence of multinucleated giant cells (MNGCs) around HCCP implants support this interpretation. These MNGCs were observed consistently across multiple samples by 28 days (Figure 7B). Importantly, MNGCs in this context are not indicative of chronic rejection but of a remodeling phenotype often associated with successful tissue integration [38,39].

4.3. Role of Multinucleated Giant Cells in Scaffold Resorption

MNGCs arise from the fusion of macrophages in response to large or persistent biomaterial interfaces. While often associated with foreign body responses, MNGCs also participate in physiological remodeling by secreting matrix metalloproteinases and acidic enzymes that facilitate scaffold resorption [40]. In HCCP implants, the controlled presence of MNGCs, in the absence of necrosis or fibrotic encapsulation, suggests a balanced remodeling activity. Their spatial association with scaffold material and lack of surrounding leukocyte infiltration suggests a regulated, non-pathologic interface.

This contrasts with HA/β-TCP, where transient inflammation and degradation byproducts may recruit inflammatory neutrophils and monocytes that are less conducive to stable vascularization [41]. The relative absence of MNGCs in HA/β-TCP sections further underscores the material-dependent variation in immune modulation and long-term scaffold remodeling.

4.4. Implant Volume Dynamics Reflect Tissue Ingrowth

The observed increase in HCCP implant volume over 28 days suggests active tissue ingrowth and extracellular matrix (ECM) deposition within the scaffold (Figure 2). Polymeric scaffolds such as HCCP can imbibe interstitial fluid and swell, providing a hydrated matrix that promotes cell survival and migration [42]. This expansion likely reflects both passive fluid absorption and active matrix deposition by infiltrating fibroblasts and endothelial cells. Quantitative volumetric analysis confirmed a significant increase in HCCP size from 3 to 28 days, contrasting with the progressive resorption and collapse of HA/β-TCP implants.

Swelling-induced volumetric changes are often associated with favorable remodeling outcomes and are particularly useful in creating pro-angiogenic niches. ECM proteins such as fibronectin and collagen, deposited during tissue ingrowth, provide binding sites for integrins on endothelial cells and enhance VEGF responsiveness [43]. Thus, HCCP’s swelling behavior may be mechanistically linked to its enhanced angiogenesis.

4.5. Temporal Dynamics of Angiogenesis

Angiogenesis in biomaterials typically follows a biphasic temporal pattern: an early phase of endothelial cell recruitment and a later phase of capillary lumenization and stabilization. The absence of vascular structures at 3 days post-implantation in both groups is consistent with this known delay. However, at 14 and 28 days, HCCP implants exhibited markedly higher vessel densities (Figure 5), consistent with the time course of neovessel formation [43].

Quantified fractional vascular area measurements further corroborated this timeline, with HCCP increasing from 0.0777 at 14 days to 0.1262 at 28 days. HA/β-TCP showed minimal gains during this period, remaining near baseline levels. These results suggest that HCCP creates a permissive microenvironment for sustained angiogenesis, in contrast to the more transient and limited response observed in HA/β-TCP.

4.6. Limitations

Although this study provides compelling evidence that HCCP enhances vascularization compared to HA/β-TCP, several limitations should be acknowledged. First, the small sample size (n = 12) and use of a single rodent model restrict generalizability, particularly to human clinical settings and load-bearing bone environments. Second, the subcutaneous implantation site represents an ectopic model that does not fully recapitulate the mechanical, cellular, and biochemical milieu of orthotopic bone defects. Third, the relatively short observation period of 28 days captures only early vascularization and immune responses, leaving longer-term remodeling, degradation kinetics, and functional bone integration unaddressed. Fourth, the placement of multiple implants within each animal using a paired contralateral design, together with routine postoperative analgesics and antibiotics, may have influenced local inflammatory or angiogenic responses. These factors were applied consistently across all study groups and timepoints and are unlikely to account for the implant-dependent differences observed, although their influence cannot be entirely excluded. Finally, the study relied on histological and gross morphological endpoints without incorporating quantitative perfusion assays or molecular analyses of angiogenic signaling, which would provide deeper mechanistic insight. Collectively, these limitations underscore the need for expanded, orthotopic, and longer-term studies to validate the translational potential of HCCP.

4.7. Future

These findings highlight the utility of HCCP as a pro-angiogenic BGS suitable for applications where rapid revascularization is essential including large defects, irradiated bone beds, or osteoporotic fractures. Its favorable porosity, degradation kinetics, and immune compatibility represent a triad of design parameters essential for next-generation tissue scaffolds. Compared with biomaterials based on HA/β-TCP [44], poly-ε-caprolactone [45], ceramic scaffolds [46], or hydroxyapatite-based composites [47], HCCP is designed to mimic aspects of glycan-mediated extracellular matrix function, which play an important role in tissue repair and regeneration. This glycan-inspired chemistry may provide a distinct microenvironment for growth factor interaction, cellular infiltration, and vascular maturation, complementing existing scaffold strategies.

Future investigations should evaluate HCCP in orthotopic defect models under load-bearing conditions and in combination with osteoinductive agents or stem cells. Quantitative studies of endothelial and pericyte recruitment, vascular lumen maturation, and perfusion are warranted to delineate the precise mechanisms underlying its vascular performance.

5. Conclusions

This study demonstrates that HCCP significantly outperforms HA/β-TCP in promoting vascularization and supporting biocompatible tissue integration in vivo. Enhanced neovascularization in HCCP implants was associated with its highly porous architecture, minimal inflammatory response, and the presence of multinucleated giant cells, which may contribute to scaffold remodeling without eliciting chronic inflammation. In contrast, HA/β-TCP elicited a pronounced immune response and showed limited vascularization over time. Longer implantation studies will be necessary to fully elucidate long-term bone regeneration and/or remodeling. These findings highlight the critical role of scaffold design in modulating host response and underscore HCCP’s potential as a next-generation BGS for applications requiring rapid vascular integration and minimal inflammatory interference.